Quinazoline-pyridine derivatives for the treatment of cancer-related disorders

ABSTRACT

Compounds of Formula I that inhibit at least one of the A2A and A2B adenosine receptors, and compositions containing compounds of Formula I and methods for synthesizing compounds of Formula I, are described herein.Also described are the use of such compounds and compositions for the treatment of a diverse array of diseases, disorders, and conditions, including cancer- and immune-related disorders that are mediated, at least in part, by the adenosine A2A receptor and/or the adenosine A2B receptor.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is the U.S. National Stage Entry under § 371 ofInternational Application No. PCT/US2018/030909, filed May 3, 2018,which claims the benefit priority to U.S. Provisional Application No.62/502,391 filed May 5, 2017 and U.S. Provisional Application No.62/624,273 filed Jan. 31, 2018, each of which is herein incorporated byreference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not Applicable

BACKGROUND OF THE INVENTION

Adenosine is a purine nucleoside compound comprising a complex ofadenine and a ribose sugar molecule (ribofuranose). Adenosine occursnaturally in mammals and plays important roles in several biochemicalprocesses, including energy transfer (as adenosine triphosphate andadenosine monophosphate) and signal transduction (as cyclic adenosinemonophosphate). Adenosine also serves in processes associated withvasodilation, including cardiac vasodilation, and acts as aneuromodulator (e.g., it is thought to be involved in promoting sleep).In addition to its involvement in these biochemical processes, adenosineis used as a therapeutic antiarrhythmic agent to treat, for example,supraventricular tachycardia. As discussed further herein, tumors evadehost responses by inhibiting immune function and promoting tolerance,and adenosine has been shown to play an important role in mediatingtumor evasion of the immune system. Adenosine signaling through A_(2A)Rsand A_(2B)Rs, expressed on a variety of immune cell subsets andendothelial cells, has been established as having an important role inprotecting tissues during inflammatory responses. As such, under certainconditions adenosine protects tumors from immune destruction (see, e.g.,Fishman, P, et al. (2009) Handb Exp Pharmacol 193:399-441).

The adenosine receptors are a class of purinergic G protein-coupledreceptors with adenosine as the endogenous ligand. The four types ofadenosine receptors in humans are referred to as A₁, A_(2A), A_(2B) andA₃. Modulation of A₁ has been proposed for the management and treatmentof, for example, neurological disorders, asthma, and heart and renalfailure; A_(2A) antagonists have been proposed for the management andtreatment of, for example, Parkinson's disease; modulation of A_(2B) hasbeen proposed for the management and treatment of, for example, chronicpulmonary diseases, including asthma; and modulation of A₃ has beenproposed for the management and treatment of, for example, asthma andchronic obstructive pulmonary diseases, glaucoma, cancer, and stroke.

Historically, modulators of adenosine receptors have been nonselective.This is acceptable in certain indications, such as where the endogenousagonist adenosine, which acts on all four adenosine receptors in cardiactissue, is administered parenterally for the treatment of severetachycardia. However, the use of sub-type selective adenosine receptoragonists and antagonists provides the potential for achieving desiredoutcomes while minimizing or eliminating adverse effects.

As such, there is a need in the art for sub-type selective adenosinereceptor agonists. The present invention addresses this need andprovides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to compounds that modulate the adenosineA_(2A) receptor (A_(2A)R) and/or the adenosine A_(2B) receptor(A_(2B)R), and compositions (e.g., pharmaceutical compositions)comprising the compounds. Such compounds, including methods of theirsynthesis, and compositions are described in detail below.

The present invention also relates to the use of such compounds andcompositions for the treatment and/or prevention of a diverse array ofdiseases, disorders and conditions mediated, in whole or in part, by theadenosine A_(2A) receptor (A_(2A)R) and/or the adenosine A_(2B) receptor(A_(2B)R). Such diseases, disorders and conditions are described indetail elsewhere herein. Unless otherwise indicated, when uses of thecompounds of the present invention are described herein, it is to beunderstood that such compounds may be in the form of a composition(e.g., a pharmaceutical composition).

As discussed hereafter, although the compounds of the present inventionare believed to affect their activity by inhibition of the adenosineA_(2A) receptor (A_(2A)R) and/or the adenosine A_(2B) receptor(A_(2B)R), a precise understanding of the compounds' underlyingmechanism of action is not required to practice the invention. It isenvisaged that the compounds may alternatively affect their activitythrough direct or indirect inhibition of adenylyl cyclase. It is alsoenvisaged that the compounds may affect their activity throughinhibition of both A_(2A) receptor (A_(2A)R) and/or the adenosine A_(2B)receptor (A_(2B)R) as well as adenylyl cyclase. Although the compoundsof the invention are generally referred to herein as adenosine A_(2A)receptor (A_(2A)R) and/or the adenosine A_(2B) receptor (A_(2B)R)inhibitors, it is to be understood that the term “A_(2A)R/A_(2B)Rinhibitors” encompasses compounds that act individually throughinhibition of A_(2A)R, A_(2B)R or adenylyl cyclase, and/or compoundsthat act through inhibition of A_(2A)R, A_(2B)R, and adenylyl cyclase.

The A_(2A) and A_(2B) cell surface adenosine receptors are found to beupregulated in various tumor cells. Thus, antagonists of the A_(2A)and/or A_(2B) adenosine receptors represent a new class of promisingoncology therapeutics.

Activation of the A_(2A) adenosine receptor results in inhibition of theimmune response to tumors via suppression of T regulatory cell functionand inhibition of natural killer cell cytotoxicity and tumor-specificCD4+/CD8+ activity. Therefore, inhibition of this receptor subtype byspecific antagonists may enhance immunotherapeutics in cancer therapy.Activation of the A_(2B) adenosine receptor plays a role in thedevelopment of tumors via upregulation of the expression levels ofangiogenic factors in microvascular endothelial cells. [See, e.g., P.Fishman et al., Handb Exp Pharmacol (2009);193:399-441]. Moreover,adenosine receptor 2A blockade has been shown to increase the efficacyof anti-PD-1 through enhanced anti-tumor T cell responses (P. Beavis, etal., Cancer Immunol Res DOI: 10.1158/2326-6066.CIR-14-0211 Published 11Feb. 2015). A more comprehensive discussion of the roles of the A_(2A)Rsand the A_(2B)Rs is set forth hereafter.

Adenosine 2A Receptor (A2AR)

The A_(2A)R (also referred to as ADORA2A) is a G protein-coupledreceptor (GPCR), family members of which possess seven transmembranealpha helices. Based on its crystallographic structure, the A_(2A)Rcomprises a ligand binding pocket distinct from that of otherstructurally determined GPCRs (e.g., the beta-2 adrenergic receptor).

As set forth elsewhere herein, adenosine is involved in mediating tumorevasion of the immune system. The A_(2A)R plays a critical, nonredundantrole in mediating adenosine-induced anti-inflammatory responses. TheA_(2A)R negatively regulates immune responses, and thus pharmacologicinhibition of A_(2A)R activation has been demonstrated to be a viablemeans of enhancing immunotherapy.

As noted above, activation of the A_(2A)R impacts the adaptive immuneresponse; by way of example, the A_(2A)R protects the host fromexcessive tissue destruction by not only acutely inhibiting T-cellfunction, but by also promoting the development of regulatory T cells.Because A_(2A)R activation is a potent inhibitor of adaptive immuneresponses, tumor-derived adenosine has been implicated in blockingantitumor immunity.

In addition to its other roles, the A_(2A)R has been implicated inselectively enhancing anti-inflammatory cytokines, promoting theupregulation of PD-1 and CTLA-4, promoting the generation of LAG-3 andFoxp3+ regulatory T cells, and mediating the inhibition of regulatory Tcells. PD-1, CTLA-4 and other immune checkpoints are discussed furtherherein. As all of these immunosuppressive properties have beenidentified as mechanisms by which tumors evade host responses, a cancerimmunotherapeutic regimen that includes an A_(2A)R antagonist may resultin enhanced tumor immunotherapy. See generally, Naganuma, M., et al.(2006) J Immunol 177:2765-769.

A_(2A)R antagonists likely play an important role in chemotherapy andradiation therapy. Mechanistically, the concomitant administration ofA_(2A)R antagonists during chemotherapy or radiation therapy has beenproposed to lead to the expansion of tumor-specific T cells whilesimultaneously preventing the induction of tumor-specific regulatory Tcells. Furthermore, combining A_(2A)R antagonists with tumor vaccines isthought to provide at least an additive effect in view of theirdivergent mechanisms of action. Finally, A_(2A)R antagonists may mosteffectively be used in combination with tumor vaccines and othercheckpoint blockers. By way of example, blocking PD-1 engagement as wellas inhibiting the A_(2A)R might mitigate the ability of tumors to turnoff tumor-specific effector T cells (see, e.g., Fishman, P, et al.(2009) Handb Exp Pharmacol 193:399-441). Moreover, adenosine signalingthrough the A_(2A)R receptor has been found to be a promising negativefeedback loop, and preclinical studies have confirmed that blockade ofA_(2A)R activation can markedly enhance anti-tumor immunity (Sitkovsky,M V, et al. (2014) Cancer Immun Res 2:598-605).

Adenosine 2B Receptor (A2BR)

The A_(2b)R (also referred to as ADORA2B) is a GPCR found in manydifferent cell types. It requires higher concentrations of adenosine foractivation than other adenosine receptor subtypes (e.g., A₁R, A_(2A)R,and A₃R) (Fredholm B B, et al. (2001) Biochem Pharmacol 61:443-448).Such conditions have been seen in, for example, tumors where hypoxia iscommonly observed. Contrary to the other adenosine receptor subtypes,the A_(2B)R may play an important role in pathophysiological conditionsassociated with massive adenosine release. Thus, selective blockade orstimulation of this adenosine receptor subtype may not interfere withthe numerous important physiological functions of adenosine mediated viaother adenosine receptor subtypes. However, the pathway leading toA_(2B)R-mediated inhibition is not fully understood.

Angiogenesis represents a pivotal mechanism for tumor growth. Theangiogenesis process is highly regulated by an array of angiogenicfactors and is triggered by adenosine under particular circumstancesthat are associated with hypoxia. The A_(2B)R is expressed in humanmicrovascular endothelial cells, where it plays an important role in theregulation of the expression of angiogenic factors such as vascularendothelial growth factor (VEGF). In certain tumor types, hypoxia hasbeen observed to cause an upregulation of A_(2B)Rs, suggesting thatA_(2B)Rs play a critical role in mediating the effects of adenosine onangiogenesis. Thus, blockade of A_(2B)Rs may limit tumor growth bylimiting the oxygen supply to the tumor cells. Furthermore, experimentsinvolving adenylate cyclase activation indicate that A_(2B)Rs are thesole adenosine receptor subtype in certain tumor cells, suggesting thatA_(2B)R antagonists may exhibit effects on particular tumor types (see,e.g., Feoktistov, I. et al. (2003) Circ Res 92:485-492).

Recent data complicate an understanding of the precise role of A_(2B)Rmodulators. As discussed above, data confirm that A_(2B)Rs play animportant role in mediating the effects of adenosine on tumor growth andprogression. Indeed, inhibition of angiogenesis and inhibition of ERK1/2 phosphorylation represent the most interesting effects for apotential anticancer treatment based on A_(2B)R as a target. However,while inhibition of angiogenesis requires the use of A_(2B)Rantagonists, inhibition of growth signaling via other clinicallyrelevant pathways (e.g., the MAP kinase pathway) might be achievedthrough treatment with A_(2B)R agonists (see, e.g., Graham, S. et al.(2001) Eur J Pharmaol 420:19-26). The results of additionalexperimentation may indicate that both agonists and antagonists willprovide useful options for treatment in combination with othertherapeutic measures if used at different stages of the disease and itstreatment.

In one particular aspect, provided herein are compounds having Formula(I):

-   or a pharmaceutically acceptable salt, hydrate, or solvate thereof,    wherein,-   the subscript m is 0 or 1, indicating a pyridine when m is 0 or a    pyridine N-oxide when m is 1;-   G¹ is N or CR^(3a);-   G² is N or CR^(3b);-   R^(3a) and R^(3b) are each independently H or C₁₋₃ alkyl;-   R^(1a) and R^(1b) are each independently selected from the group    consisting of    -   i) H    -   ii) C₁₋₈ alkyl optionally substituted with from 1-3 R⁵        substituents,    -   iii) —X¹—O—C₁₋₈ alkyl optionally substituted with from 1-3 R⁵        substituents,    -   iv) —C(O)—R⁶,    -   v) Y optionally substituted with 1-3 R⁷ substituents, and    -   vi) —X¹—Y optionally substituted with 1-3 R⁷ substituents; or    -   vii) R^(1a) and R^(1b) together with the nitrogen to which they        are attached form a 5-6 membered heterocycloalkyl ring        optionally substituted with from 1-3 R⁸ substituents,    -   wherein the heterocycloalkyl has 0-2 additional heteroatom ring        vertices selected from the group consisting of O, N, and S;-   each Y is C₃₋₈ cycloalkyl or 4 to 6-membered heterocycloalkyl having    1-3 heteroatom ring vertices selected from the group consisting of    O, N, and S;-   R² and R⁴ are each independently H or C₁₋₃ alkyl;-   each X¹ is C₁₋₆ alkylene;-   each R⁵ is independently selected from the group consisting of    hydroxyl, C₃₋₈ cycloalkyl, phenyl, —O-phenyl, —C(O)OR^(a) and oxo;-   each R⁶ is C₁₋₈ alkyl or Y, each of which is optionally substituted    with 1-3 substituents selected from the group consisting of    hydroxyl, —O-phenyl, phenyl, and —O—C₁₋₈ alkyl;-   each R⁷ is independently selected from the group consisting of C₁₋₈    alkyl, hydroxyl, —O—C₁₋₈ alkyl, oxo, and C(O)OR^(a);-   each R⁸ is independently selected from the group consisting of C₁₋₈    alkyl, hydroxyl, and oxo;-   the subscript n is 0, 1, 2 or 3;-   each R⁹ is independently selected from the group consisting of C₁₋₈    alkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl,    —X¹—O—X¹—O—C₁₋₈ alkyl, —C(O)OR^(a), halogen, cyano, —NR^(b)R^(c), Y,    —X¹—C₃₋₈ cycloalkyl, and —X²—Z, wherein X² is selected from the    group consisting of C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C₁₋₄    alkylene-O—C₁₋₄ alkylene-, —C(O)—, and —S(O)₂—, Z is 4 to 6-membered    heterocycloalkyl having 1-3 heteroatom ring vertices selected from    the group consisting of O, N, and S, and wherein each of said R⁹    substituents is optionally substituted with 1-3 R¹¹;-   each of R^(10a), R^(10b), R^(10c) and R^(10d) is independently    selected from the group consisting of H, C₁₋₈ alkyl, halo, cyano,    —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl, —S(O)₂—C₁₋₆    alkyl, —C(O)NR^(d)R^(e), and 4-6-membered heteroaryl having from 1-3    heteroatom ring vertices selected from the group consisting of O, N,    and S, wherein each of said R^(10a-d) substituents is optionally    substituted with 1-3 R¹², or two of R^(10a), R^(10b), R^(10c) and    R^(10d) on adjacent ring vertices are optionally combined to form a    5-membered heterocyclic ring optionally substituted with 1-2    halogens;-   each R¹¹ is independently selected from the group consisting of    hydroxyl, oxo, halo, cyano, —NR^(d)R^(e), —C(O)OR^(a), phenyl, C₃₋₈    cycloalkyl, and C₁₋₄ alkyl optionally substituted with —C(O)OR^(a);-   each R¹² is independently selected from the group consisting of    halo, cyano, hydroxy, —C(O)OR^(a); and-   each R^(a) is H or C₁₋₆ alkyl;-   each R^(b) and R^(c) are independently selected from the group    consisting of H, C₁₋₈ alkyl, —S(O)₂—C₁₋₆ alkyl, —C(O)OR^(a), and    —X¹—C(O)OR^(a); and-   each R^(d) and R^(e) are independently selected from the group    consisting of H, C₁₋₈ alkyl, —S(O)₂—C₁₋₆ alkyl.

In some embodiments, provided herein are methods for treating orpreventing cancer in a subject (e.g., a human) comprising administeringto the subject a therapeutically effective amount of at least oneA_(2A)R/A_(2B)R inhibitor described herein. In some embodiments,provided herein are methods of treating or preventing a cancer in asubject by administering to the subject at least one of the compoundsdescribed herein in an amount effective to reverse or stop theprogression of A_(2A)R-mediated immunosuppression. In some embodiments,the A_(2A)R-mediated immunosuppression is mediated by anantigen-presenting cell (APC).

Examples of the cancers that may be treated using the compounds andcompositions described herein include, but are not limited to: cancersof the prostate, colorectum, pancreas, cervix, stomach, endometrium,brain, liver, bladder, ovary, testis, head, neck, skin (includingmelanoma and basal carcinoma), mesothelial lining, white blood cell(including lymphoma and leukemia) esophagus, breast, muscle, connectivetissue, lung (including small-cell lung carcinoma and non-small-celllung carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma,mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma,choriocarcinoma, cutaneous basocellular carcinoma, and testicularseminoma. In some embodiments of the present invention, the cancer ismelanoma, colon cancer, pancreatic cancer, breast cancer, prostatecancer, lung cancer, leukemia, a brain tumor, lymphoma, sarcoma, ovariancancer, head and neck cancer, cervical cancer or Kaposi's sarcoma.Cancers that are candidates for treatment with the compounds andcompositions of the present invention are discussed further hereafter.

Also provided herein are methods of treating a subject receiving a bonemarrow transplant or peripheral blood stem cell transplant byadministering a therapeutically effective amount of an A_(2A)R/A_(2B)Rinhibitor sufficient to increase the delayed-type hypersensitivityreaction to tumor antigen, delay the time-to-relapse of post-transplantmalignancy, increase relapse-free survival time post-transplant, and/orincrease long-term post-transplant survival.

In certain embodiments, provided herein are methods for treating orpreventing an infective disorder (e.g., a viral infection) in a subject(e.g., a human) comprising administering to the subject atherapeutically effective amount of at least one A_(2A)R/A_(2B)Rinhibitor (e.g., a novel inhibitor of the instant invention). In someembodiments, the infective disorder is a viral infection (e.g., achronic viral infection), a bacterial infection, a fungal infection, ora parasitic infection. In certain embodiments, the viral infection ishuman immunodeficiency virus or cytomegalovirus.

In still other embodiments, provided herein are methods for treating orpreventing an immune-related disease, disorder or condition in a subject(e.g., a human), comprising administering to the subject atherapeutically effective amount of at least one A_(2A)R/A_(2B)Rinhibitor described herein. Examples of immune-related diseases,disorders and conditions are described hereafter.

Other diseases, disorders and conditions that can be treated orprevented, in whole or in part, by modulation of A_(2A)R/A_(2B)Ractivity are candidate indications for the A_(2A)R/A_(2B)R inhibitorcompounds as provided herein.

Also provided herein is the use of the described A_(2A)R/A_(2B)Rinhibitors in combination with one or more additional agents. The one ormore additional agents may have some adenosine A_(2A) receptor and/oradenosine A_(2B) receptor modulating activity; alternatively, they mayfunction through distinct mechanisms of action. In some embodiments,such agents comprise radiation (e.g., localized radiation therapy ortotal body radiation therapy) and/or other treatment modalities of anon-pharmacological nature. When combination therapy is utilized, thecompound(s) described herein and the one additional agent(s) may be inthe form of a single composition or multiple compositions, and thetreatment modalities may be administered concurrently, sequentially, orthrough some other regimen. By way of example, the present inventioncontemplates a treatment regimen wherein a radiation phase is followedby a chemotherapeutic phase. The combination therapy may have anadditive or synergistic effect. Other benefits of combination therapyare described hereafter.

In particular embodiments, provided herein are methods wherein theA_(2A)R/A_(2B)R inhibitors described herein are used in combination withimmune checkpoint inhibitors. The blockade of immune checkpoints, whichresults in the amplification of antigen-specific T cell responses, hasbeen shown to be a promising approach in human cancer therapeutics.Examples of immune checkpoints (ligands and receptors), some of whichare selectively upregulated in various types of tumor cells, that arecandidates for blockade include PD1 (programmed cell death protein 1);PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4(cytotoxic T-lymphocyte associated antigen 4); TIM3 (T-cell membraneprotein 3); LAG3 (lymphocyte activation gene 3); TIGIT (T cellimmunoreceptor with Ig and ITIM domains); and Killer InhibitoryReceptors. Immune checkpoint inhibitors, and combination therapytherewith, are discussed in detail elsewhere herein.

In other embodiments, provided herein are methods for treating cancer ina subject, comprising administering to the subject a therapeuticallyeffective amount of at least one A_(2A)R/A_(2B)R inhibitor and at leastone chemotherapeutic agent, such agents including, but not limited toalkylating agents (e.g., nitrogen mustards such as chlorambucil,cyclophosphamide, isofamide, mechlorethamine, melphalan, and uracilmustard; aziridines such as thiotepa; methanesulphonate esters such asbusulfan; nucleoside analogs (e.g., gemcitabine); nitroso ureas such ascarmustine, lomustine, and streptozocin; topoisomerase 1 inhibitors(e.g., irinotecan); platinum complexes such as cisplatin, carboplatinand oxaliplatin; bioreductive alkylators such as mitomycin,procarbazine, dacarbazine and altretamine); anthracycline-basedtherapies (e.g., doxorubicin, daunorubicin, epirubicin and idarubicin);DNA strand-breakage agents (e.g., bleomycin); topoisomerase IIinhibitors (e.g., amsacrine, dactinomycin, daunorubicin, idarubicin,mitoxantrone, doxorubicin, etoposide, and teniposide); DNA minor groovebinding agents (e.g., plicamydin); antimetabolites (e.g., folateantagonists such as methotrexate and trimetrexate; pyrimidineantagonists such as fluorouracil, fluorodeoxyuridine, CB3717,azacitidine, cytarabine, and floxuridine; purine antagonists such asmercaptopurine, 6-thioguanine, fludarabine, pentostatin; asparginase;and ribonucleotide reductase inhibitors such as hydroxyurea); tubulininteractive agents (e.g., vincristine, estramustine, vinblastine,docetaxol, epothilone derivatives, and paclitaxel); hormonal agents(e.g., estrogens; conjugated estrogens; ethinyl estradiol;diethylstilbesterol; chlortrianisen; idenestrol; progestins such ashydroxyprogesterone caproate, medroxyprogesterone, and megestrol; andandrogens such as testosterone, testosterone propionate,fluoxymesterone, and methyltestosterone); adrenal corticosteroids (e.g.,prednisone, dexamethasone, methylprednisolone, and prednisolone);leutinizing hormone releasing agents or gonadotropin-releasing hormoneantagonists (e.g., leuprolide acetate and goserelin acetate); andantihormonal antigens (e.g., tamoxifen, antiandrogen agents such asflutamide; and antiadrenal agents such as mitotane andaminoglutethimide). The present invention also contemplates the use ofthe A_(2A)R/A_(2B)R inhibitors in combination with other agents known inthe art (e.g., arsenic trioxide) and other chemotherapeutic agentsdeveloped in the future.

In some embodiments, provided herein are methods of treating cancer inwhich a therapeutically effective amount of an A_(2A)R/A_(2B)R inhibitordescribed herein is administered in combination with at least onechemotherapeutic agent, resulting in a cancer survival rate greater thanthe cancer survival rate observed by administering either alone. Infurther embodiments drawn to methods of treating cancer, theadministration of a therapeutically effective amount of anA_(2A)R/A_(2B)R inhibitor described herein in combination with at leastone chemotherapeutic agent results in a reduction of tumor size or aslowing of tumor growth greater than reduction of the tumor size ortumor growth observed by administration of one agent alone.

In further embodiments, the present invention contemplates methods fortreating or preventing cancer in a subject, comprising administering tothe subject a therapeutically effective amount of at least oneA_(2A)R/A_(2B)R inhibitor described herein and at least one signaltransduction inhibitor (STI). In a particular embodiment, the at leastone STI is selected from the group consisting of bcr/abl kinaseinhibitors, epidermal growth factor (EGF) receptor inhibitors, her-2/neureceptor inhibitors, and farnesyl transferase inhibitors (FTIs). Othercandidate STI agents are set forth elsewhere herein.

The present invention also contemplates methods of augmenting therejection of tumor cells in a subject comprising administering anA_(2A)R/A_(2B)R inhibitor in conjunction with at least onechemotherapeutic agent and/or radiation therapy, wherein the resultingrejection of tumor cells is greater than that obtained by administeringeither the A_(2A)R/A_(2B)R inhibitor, the chemotherapeutic agent or theradiation therapy alone.

In further embodiments, the present invention provides methods fortreating cancer in a subject, comprising administering to the subject atherapeutically effective amount of at least one A_(2A)R/A_(2B)Rinhibitor and at least one immunomodulator other than an A_(2A)R/A_(2B)Rinhibitors. In particular embodiments, the at least one immunomodulatoris selected from the group consisting of CD4OL, B7, B7RP1, ant-CD40,anti-CD38, anti-ICOS, 4-IBB ligand, dendritic cell cancer vaccine, IL2,IL12, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC,IFN-a/-13, M-CSF, IL-3, GM-CSF, IL-13, anti-IL-10 and indoleamine2,3-dioxygenase 1 (IDO1). Other candidate immunomodulator agents are setforth elsewhere herein.

The present invention contemplates embodiments comprising methods fortreating or preventing an infective disorder (e.g., a viral infection)in a subject (e.g., a human) comprising administering to the subject atherapeutically effective amount of at least one A_(2A)R/A_(2B)Rinhibitor described herein and a therapeutically effective amount of ananti-infective agent(s).

In some embodiments of the present invention, the additional therapeuticagent is a cytokine, including, for example granulocyte-macrophagecolony stimulating factor (GM-CSF) or flt3-ligand. The present inventionalso contemplates methods for treating or preventing a viral infection(e.g., a chronic viral infection) including, but not limited to,hepatitis C virus (HCV), human papilloma virus (HPV), cytomegalovirus(CMV), Epstein-Barr virus (EBV), varicella zoster virus, coxsackievirus, and human immunodeficiency virus (HIV). The use of the compoundsdescribed herein to treat (either alone or as a component of combinationtherapy) infection is discussed further hereafter.

In additional embodiments, treatment of an infective disorder iseffected through the co-administration of a vaccine in combination withadministration of a therapeutically effective amount of anA_(2A)R/A_(2B)R inhibitor of the present invention. In some embodiments,the vaccine is an anti-viral vaccine, including, for example, ananti-HIV vaccine. In other embodiments, the vaccine is effective againsttuberculosis or malaria. In still other embodiments, the vaccine is atumor vaccine (e.g., a vaccine effective against melanoma); the tumorvaccine may comprise genetically modified tumor cells or a geneticallymodified cell line, including genetically modified tumor cells or agenetically modified cell line that has been transfected to expressgranulocyte-macrophage stimulating factor (GM-C SF). In particularembodiments, the vaccine includes one or more immunogenic peptidesand/or dendritic cells.

In some embodiments, the present invention contemplates methods of usingthe compounds described herein in combination with one or moreantimicrobial agents.

In certain embodiments drawn to treatment of an infection byadministering an A_(2A)R/A_(2B)R inhibitor and at least one additionaltherapeutic agent, a symptom of infection observed after administeringboth the A_(2A)R/A_(2B)R inhibitor and the additional therapeutic agentis improved over the same symptom of infection observed afteradministering either alone. In some embodiments, the symptom ofinfection observed can be reduction in viral load, increase in CD4⁺ Tcell count, decrease in opportunistic infections, increased survivaltime, eradication of chronic infection, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

NOT APPLICABLE

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is further described, it is to beunderstood that the invention is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. It is furthernoted that the claims may be drafted to exclude any optional element. Assuch, this statement is intended to serve as antecedent basis for use ofsuch exclusive terminology such as “solely,” “only” and the like inconnection with the recitation of claim elements, or use of a “negative”limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Further,the dates of publication provided may be different from the actualpublication dates, which may need to be independently confirmed.

General

Provided herein, for example, are compounds and compositions forinhibition of the adenosine A_(2A) receptor (A_(2A)R) and/or theadenosine A_(2B) receptor (A_(2B)R), and pharmaceutical compositionscomprising the same. Also provided herein are, for example, methods oftreating or preventing a disease, disorder or condition, or a symptomthereof, mediated by inhibition of adenosine A_(2A) receptor (A_(2A)R)and/or the adenosine A_(2B) receptor (A_(2B)R).

Definitions

Unless otherwise indicated, the following terms are intended to have themeaning set forth below. Other terms are defined elsewhere throughoutthe specification.

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical, having the number of carbon atoms designated (i.e. C₁₋₈ meansone to eight carbons). Alkyl can include any number of carbons, such asC₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈, C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅,C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ and C₅₋₆. Examples of alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

The term “alkylene” refers to a straight or branched, saturated,aliphatic radical having the number of carbon atoms indicated, andlinking at least two other groups, i.e., a divalent hydrocarbon radical.The two moieties linked to the alkylene can be linked to the same atomor different atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is 1, 2, 3,4, 5 or 6. Representative alkylene groups include, but are not limitedto, methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene. Alkylene groups, often referred toas X¹ or X² groups in the present application, can be substituted orunsubstituted. When a group comprising X¹ or X² is optionallysubstituted, it is understood that the optional substitutions may be onthe alkylene portion of the moiety.

The term “cycloalkyl” refers to hydrocarbon rings having the indicatednumber of ring atoms (e.g., C₃₋₆ cycloalkyl) and being fully saturatedor having no more than one double bond between ring vertices.“Cycloalkyl” is also meant to refer to bicyclic and polycyclichydrocarbon rings such as, for example, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, etc. In some embodiments, the cycloalkyl compoundsof the present disclosure are monocyclic C₃₋₆ cycloalkyl moieties.

The term “heterocycloalkyl” refers to a cycloalkyl ring having theindicated number of ring vertices (or members) and having from one tofive heteroatoms selected from N, O, and S, which replace one to five ofthe carbon vertices, and wherein the nitrogen and sulfur atoms areoptionally oxidized, and the nitrogen atom(s) are optionallyquaternized. The cycloheteroalkyl may be a monocyclic, a bicyclic or apolycylic ring system. Non limiting examples of cycloheteroalkyl groupsinclude pyrrolidine, imidazolidine, pyrazolidine, butyrolactam,valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide,piperidine, 1,4-dioxane, morpholine, thiomorpholine,thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran,pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran,tetrhydrothiophene, quinuclidine, and the like. A cycloheteroalkyl groupcan be attached to the remainder of the molecule through a ring carbonor a heteroatom.

As used herein, a wavy line, “

”, that intersects a single, double or triple bond in any chemicalstructure depicted herein, represent the point attachment of the single,double, or triple bond to the remainder of the molecule. Additionally, abond extending to the center of a ring (e.g., a phenyl ring) is meant toindicate attachment at any of the available ring vertices. One of skillin the art will understand that multiple substituents shown as beingattached to a ring will occupy ring vertices that provide stablecompounds and are otherwise sterically compatible. For a divalentcomponent, a representation is meant to include either orientation(forward or reverse). For example, the group “—C(O)NH—” is meant toinclude a linkage in either orientation: —C(O)NH— or —NHC(O)—, andsimilarly, “—O—CH₂CH₂—” is meant to include both —O—CH₂CH₂— and—CH₂CH₂—O—.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“C₁₋₄ haloalkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon group which can be a single ring ormultiple rings (up to three rings) which are fused together or linkedcovalently. Non-limiting examples of aryl groups include phenyl,naphthyl and biphenyl.

The term “heteroaryl” refers to aryl groups (or rings) that contain fromone to five heteroatoms selected from N, O, and S, wherein the nitrogenand sulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofheteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimindinyl,triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl,phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl,benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl,benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl,imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl,quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl,imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl,pyrrolyl, thiazolyl, furyl, thienyl and the like. Substituents for aheteroaryl ring can be selected from the group of acceptablesubstituents described below.

The above terms (e.g., “alkyl,” “aryl” and “heteroaryl”), in someembodiments, will be optionally substituted. Selected substituents foreach type of radical are provided below.

Optional substituents for the alkyl radicals (including those groupsoften referred to as alkylene, alkenyl, and alkynyl) can be a variety ofgroups selected from: halogen, —OR′, —NR′R″, —SR′, —SiR′R″R″′, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R″′,—NR″ C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —CN (cyano), —NO₂, aryl, aryloxy,oxo, cycloalkyl and heterocycloalkyl in a number ranging from zero to (2m′+1), where m′ is the total number of carbon atoms in such radical. R′,R″ and R″′ each independently refer to hydrogen, unsubstituted C₁₋₈alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, C₁₋₈alkoxy or C₁₋₈ thioalkoxy groups, or unsubstituted aryl-C₁₋₄ alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or7-membered ring. For example, —NR′R″ is meant to include 1-pyrrolidinyland 4-morpholinyl.

Optional substituents for the cycloalkyl and heterocycloalkyl radicalscan be a variety of groups selected from: alkyl optionally substitutedwith C(O)OR′, halogen, —OR′, —NR′R″, —SR′, —SiR′R″R″′, —OC(O)R′,—C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C (O)R′, —NR′ —C(O)NR″R″′,—NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′,—S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —CN (cyano), —NO₂, aryl, aryloxy andoxo. R′, R″ and R″′ each independently refer to hydrogen, unsubstitutedC₁₋₈ alkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, C₁₋₈alkoxy or C₁₋₈ thioalkoxy groups, or unsubstituted aryl-C₁₋₄ alkylgroups.

Similarly, optional substituents for the aryl and heteroaryl groups arevaried and are generally selected from: -halogen, —OR′, —OC(O)R′,—NR′R″, —SR′, —R′, —CN, —NO₂, —CO₂R′, —CONR′R″, —C(O)R′, —OC(O)NR′R″,—NR″C(O)R′, —NR″ C(O)₂R′, —NR′—C(O)NR″R″′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NR′S(O)₂R″, —N₃,perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a number rangingfrom zero to the total number of open valences on the aromatic ringsystem; and where R′, R″ and R″′ are independently selected fromhydrogen, C₁₋₈ alkyl, C₁₋₈ haloalkyl, C₃₋₆ cycloalkyl, C₂₋₈alkenyl andC₂₋₈ alkynyl. Other suitable substituents include each of the above arylsubstituents attached to a ring atom by an alkylene tether of from 1-6carbon atoms.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula-A-(CR^(f)R^(g))_(r)—B—, wherein A and B are independently —CH₂—, —O—,—NH—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, r is an integerof from 1 to 3, and R^(f) and R^(g) are each independently H of halogen.One of the single bonds of the new ring so formed may optionally bereplaced with a double bond. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and tare independently integers of from 0 to 3, and X is —O—, —NR′—, —S—,—S(O)—, —S(O)₂—, or —S(O)₂NR′—. The substituent R′ in —NR′— and—S(O)₂NR′— is selected from hydrogen or unsubstituted C₁₋₆ alkyl.

As used herein, the term “heteroatom” is meant to include oxygen (O),nitrogen (N), sulfur (S) and silicon (Si).

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention. Inaddition to salt forms, the present invention provides compounds whichare in a prodrug form. Prodrugs of the compounds described herein arethose compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent. Prodrugs aredescribed in more detail elsewhere herein.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention may be present, underparticular conditions, as polymorphs. Polymorphism refers to the abilityof a solid material to exist in more than one crystal structure form orphase, wherein the molecules in the crystal lattice have differentarrangements or conformations. If such types of differences exist due topacking it is referred to as “packing polymorphism”, and if they existdue to differences in conformation it is referred to as “conformationalpolymorphism”. Different polymorphs of the same compound often displaydifferent physical properties, including packing properties,spectroscopic properties, thermodynamic properties, solubility, andmelting point; kinetic properties such as rate of dissolution andstability; and mechanical properties such as hardness and tensilestrength.

Polymorphs can be classified as one of two types according to theirstability with respect to different ranges of temperature and pressure.In a monotropic system, only one polymorph (i.e., monotrope) is stable,and it exhibits lower free energy content and solubility at alltemperatures and pressure below melting point. In an enantiotropicsystem, one polymorph is stable at a certain temperature and pressure,while the other polymorph(s) is stable at various temperatures andpressure.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. When a stereochemical depiction is shown, it is meantto refer the compound in which one of the isomers is present andsubstantially free of the other isomer. ‘Substantially free of’ anotherisomer indicates at least an 80/20 ratio of the two isomers, morepreferably 90/10, or 95/5 or more. In some embodiments, one of theisomers will be present in an amount of at least 99%.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. Unnatural proportions of an isotope may bedefined as ranging from the amount found in nature to an amountconsisting of 100% of the atom in question. For example, the compoundsmay incorporate radioactive isotopes, such as for example tritium (³H),iodine-125 (¹²⁵I) or carbon-14 (¹⁴C), or non-radioactive isotopes, suchas deuterium (²H) or carbon-13 (¹³C). Such isotopic variations canprovide additional utilities to those described elsewhere within thisapplication. For instance, isotopic variants of the compounds of theinvention may find additional utility, including but not limited to, asdiagnostic and/or imaging reagents, or as cytotoxic/radiotoxictherapeutic agents. Additionally, isotopic variants of the compounds ofthe invention can have altered pharmacokinetic and pharmacodynamiccharacteristics which can contribute to enhanced safety, tolerability orefficacy during treatment. All isotopic variations of the compounds ofthe present invention, whether radioactive or not, are intended to beencompassed within the scope of the present invention.

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “administration”, “administer” and the like, as they apply to,for example, a subject, cell, tissue, organ, or biological fluid, referto contact of, for example, an inhibitor of A_(2A)R/A_(2B)R, apharmaceutical composition comprising same, or a diagnostic agent to thesubject, cell, tissue, organ, or biological fluid. In the context of acell, administration includes contact (e.g., in vitro or ex vivo) of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell.

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering an inhibitor of A_(2A)R/A_(2B)R or apharmaceutical composition comprising same) initiated after a disease,disorder or condition, or a symptom thereof, has been diagnosed,observed, and the like so as to eliminate, reduce, suppress, mitigate,or ameliorate, either temporarily or permanently, at least one of theunderlying causes of a disease, disorder, or condition afflicting asubject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (e.g., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering an A_(2A)R/A_(2B)R inhibitor ora pharmaceutical composition comprising same) initiated in a manner(e.g., prior to the onset of a disease, disorder, condition or symptomthereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount capable of having any detectable, positiveeffect on any symptom, aspect, or characteristic of a disease, disorderor condition when administered to the subject. The therapeuticallyeffective amount can be ascertained by measuring relevant physiologicaleffects, and it can be adjusted in connection with the dosing regimenand diagnostic analysis of the subject's condition, and the like. By wayof example, measurement of the serum level of an A_(2A)R/A_(2B)Rinhibitor (or, e.g., a metabolite thereof) at a particular timepost-administration may be indicative of whether a therapeuticallyeffective amount has been used.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., serumconcentration) or subjective parameter (e.g., a subject's feeling ofwell-being).

The term “small molecules” refers to chemical compounds having amolecular weight that is less than about 10 kDa, less than about 2 kDa,or less than about 1 kDa. Small molecules include, but are not limitedto, inorganic molecules, organic molecules, organic molecules containingan inorganic component, molecules comprising a radioactive atom, andsynthetic molecules. Therapeutically, a small molecule may be morepermeable to cells, less susceptible to degradation, and less likely toelicit an immune response than large molecules.

The term “ligand” refers to, for example, a peptide, a polypeptide, amembrane-associated or membrane-bound molecule, or a complex thereof,that can act as an agonist or antagonist of a receptor. A ligandencompasses natural and synthetic ligands, e.g., cytokines, cytokinevariants, analogs, muteins, and binding compositions derived fromantibodies, as well as small molecules. The term also encompasses anagent that is neither an agonist nor antagonist, but that can bind to areceptor without significantly influencing its biological properties,e.g., signaling or adhesion. Moreover, the term includes amembrane-bound ligand that has been changed by, e.g., chemical orrecombinant methods, to a soluble version of the membrane-bound ligand.A ligand or receptor may be entirely intracellular, that is, it mayreside in the cytosol, nucleus, or some other intracellular compartment.The complex of a ligand and receptor is termed a “ligand-receptorcomplex.”

The terms “inhibitors” and “antagonists”, or “activators” and “agonists”refer to inhibitory or activating molecules, respectively, for example,for the activation of, e.g., a ligand, receptor, cofactor, gene, cell,tissue, or organ. Inhibitors are molecules that decrease, block,prevent, delay activation, inactivate, desensitize, or down-regulate,e.g., a gene, protein, ligand, receptor, or cell. Activators aremolecules that increase, activate, facilitate, enhance activation,sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, orcell. An inhibitor may also be defined as a molecule that reduces,blocks, or inactivates a constitutive activity. An “agonist” is amolecule that interacts with a target to cause or promote an increase inthe activation of the target. An “antagonist” is a molecule that opposesthe action(s) of an agonist. An antagonist prevents, reduces, inhibits,or neutralizes the activity of an agonist, and an antagonist can alsoprevent, inhibit, or reduce constitutive activity of a target, e.g., atarget receptor, even where there is no identified agonist.

The terms “modulate”, “modulation” and the like refer to the ability ofa molecule (e.g., an activator or an inhibitor) to increase or decreasethe function or activity of A_(2A)R/A_(2B)R, either directly orindirectly. A modulator may act alone, or it may use a cofactor, e.g., aprotein, metal ion, or small molecule. Examples of modulators includesmall molecule compounds and other bioorganic molecules. Numerouslibraries of small molecule compounds (e.g., combinatorial libraries)are commercially available and can serve as a starting point foridentifying a modulator. The skilled artisan is able to develop one ormore assays (e.g., biochemical or cell-based assays) in which suchcompound libraries can be screened in order to identify one or morecompounds having the desired properties; thereafter, the skilledmedicinal chemist is able to optimize such one or more compounds by, forexample, synthesizing and evaluating analogs and derivatives thereof.Synthetic and/or molecular modeling studies can also be utilized in theidentification of an Activator.

The “activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor; to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity; to the modulation of activities ofother molecules; and the like. The term “proliferative activity”encompasses an activity that promotes, that is necessary for, or that isspecifically associated with, for example, normal cell division, as wellas cancer, tumors, dysplasia, cell transformation, metastasis, andangiogenesis.

As used herein, “comparable”, “comparable activity”, “activitycomparable to”, “comparable effect”, “effect comparable to”, and thelike are relative terms that can be viewed quantitatively and/orqualitatively. The meaning of the terms is frequently dependent on thecontext in which they are used. By way of example, two agents that bothactivate a receptor can be viewed as having a comparable effect from aqualitative perspective, but the two agents can be viewed as lacking acomparable effect from a quantitative perspective if one agent is onlyable to achieve 20% of the activity of the other agent as determined inan art-accepted assay (e.g., a dose-response assay) or in anart-accepted animal model. When comparing one result to another result(e.g., one result to a reference standard), “comparable” frequently(though not always) means that one result deviates from a referencestandard by less than 35%, by less than 30%, by less than 25%, by lessthan 20%, by less than 15%, by less than 10%, by less than 7%, by lessthan 5%, by less than 4%, by less than 3%, by less than 2%, or by lessthan 1%. In particular embodiments, one result is comparable to areference standard if it deviates by less than 15%, by less than 10%, orby less than 5% from the reference standard. By way of example, but notlimitation, the activity or effect may refer to efficacy, stability,solubility, or immunogenicity.

“Substantially pure” indicates that a component makes up greater thanabout 50% of the total content of the composition, and typically greaterthan about 60% of the total polypeptide content. More typically,“substantially pure” refers to compositions in which at least 75%, atleast 85%, at least 90% or more of the total composition is thecomponent of interest. In some cases, the polypeptide will make upgreater than about 90%, or greater than about 95% of the total contentof the composition.

The terms “specifically binds” or “selectively binds”, when referring toa ligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated conditions, a specified ligand binds to a particularreceptor and does not bind in a significant amount to other proteinspresent in the sample. The antibody, or binding composition derived fromthe antigen-binding site of an antibody, of the contemplated methodbinds to its antigen, or a variant or mutein thereof, with an affinitythat is at least two-fold greater, at least ten times greater, at least20-times greater, or at least 100-times greater than the affinity withany other antibody, or binding composition derived therefrom. In aparticular embodiment, the antibody will have an affinity that isgreater than about 10⁹ liters/mol, as determined by, e.g., Scatchardanalysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239).

The term “response,” for example, of a cell, tissue, organ, or organism,encompasses a change in biochemical or physiological behavior, e.g.,concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming. In certaincontexts, the terms “activation”, “stimulation”, and the like refer tocell activation as regulated by internal mechanisms, as well as byexternal or environmental factors; whereas the terms “inhibition”,“down-regulation” and the like refer to the opposite effects.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like.

As used herein, the terms “variants” and “homologs” are usedinterchangeably to refer to amino acid or DNA sequences that are similarto reference amino acid or nucleic acid sequences, respectively. Theterm encompasses naturally-occurring variants andnon-naturally-occurring variants. Naturally-occurring variants includehomologs (polypeptides and nucleic acids that differ in amino acid ornucleotide sequence, respectively, from one species to another), andallelic variants (polypeptides and nucleic acids that differ in aminoacid or nucleotide sequence, respectively, from one individual toanother within a species). Thus, variants and homologs encompassnaturally occurring DNA sequences and proteins encoded thereby and theirisoforms, as well as splice variants of a protein or gene. The termsalso encompass nucleic acid sequences that vary in one or more basesfrom a naturally-occurring DNA sequence but still translate into anamino acid sequence that corresponds to the naturally-occurring proteindue to degeneracy of the genetic code. Non-naturally-occurring variantsand homologs include polypeptides and nucleic acids that comprise achange in amino acid or nucleotide sequence, respectively, where thechange in sequence is artificially introduced (e.g., muteins); forexample, the change is generated in the laboratory by human intervention(“hand of man”). Therefore, non-naturally occurring variants andhomologs may also refer to those that differ from thenaturally-occurring sequences by one or more conservative substitutionsand/or tags and/or conjugates.

The term “muteins” as used herein refers broadly to mutated recombinantproteins. These proteins usually carry single or multiple amino acidsubstitutions and are frequently derived from cloned genes that havebeen subjected to site-directed or random mutagenesis, or fromcompletely synthetic genes.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

Adenosine A_(2A) Receptor and Adenosine A_(2B) Receptor and InhibitionThereof

As set forth above, although a precise understanding of the underlyingmechanism of action by which the compounds of the present inventioneffect their activity is not required to practice the invention, thecompounds (or a subset thereof) are believed to inhibit adenosine A_(2A)receptor (A_(2A)R) and/or the adenosine A_(2B) receptor (A_(2B)R).Alternatively, the compounds (or a subset thereof) may inhibit adenylylcyclase function. The compounds (or a subset thereof) may also haveinhibitor activity on the A_(2A) receptor (A_(2A)R), the adenosineA_(2B) receptor (A_(2B)R) as well as adenylyl cyclase. Although thecompounds of the invention are generally referred to herein as adenosineA_(2A) receptor (A_(2A)R) and/or the adenosine A_(2B) receptor (A_(2B)R)inhibitors, it is to be understood that the term “A_(2A)R/A_(2B)Rinhibitors” encompasses compounds that act individually throughinhibition of A_(2A)R, A_(2B)R or adenylyl cyclase, and/or compoundsthat act through inhibition of A_(2A)R, A_(2B)R, and adenylyl cyclase.

Identification of Adenosine A_(2A) Receptor and Adenosine A_(2B)Receptor Inhibitors Possessing Desirable Characteristics

The present invention is drawn, in part, to the identification ofinhibitors of the adenosine A_(2A) receptor and/or the adenosine A_(2B)receptor with at least one property or characteristic that is oftherapeutic relevance. Candidate inhibitors may be identified by using,for example, an art-accepted assay or model, examples of which aredescribed herein.

After identification, candidate inhibitors can be further evaluated byusing techniques that provide data regarding characteristics of theinhibitors (e.g., pharmacokinetic parameters, means of determiningsolubility or stability). Comparisons of the candidate inhibitors to areference standard (which may the “best-of-class” of current inhibitors)are indicative of the potential viability of such candidates.

COMPOUNDS OF THE INVENTION

In one particular aspect, provided herein are compounds having Formula(I):

-   or a pharmaceutically acceptable salt, hydrate, or solvate thereof,    wherein,-   the subscript m is 0 or 1, indicating a pyridine when m is 0 or a    pyridine N-oxide when m is 1;-   G¹ is N or CR^(3a);-   G² is N or CR^(3b);-   R^(3a) and R^(3b) are each independently H or C₁₋₃ alkyl;-   R^(1a) and R^(1b) are each independently selected from the group    consisting of    -   i) H    -   ii) C₁₋₈ alkyl optionally substituted with from 1-3 R⁵        substituents,    -   iii) —X¹—O—C₁₋₈ alkyl optionally substituted with from 1-3 R⁵        substituents,    -   iv) —C(O)—R⁶,    -   v) Y optionally substituted with 1-3 R⁷ substituents, and    -   vi) —X¹—Y optionally substituted with 1-3 R⁷ substituents; or    -   vii) R^(1a) and R^(1b) together with the nitrogen to which they        are attached form a 5-6 membered heterocycloalkyl ring        optionally substituted with from 1-3 R⁸ substituents, wherein        the heterocycloalkyl has 0-2 additional heteroatom ring vertices        selected from the group consisting of O, N, and S;    -   each Y is C₃₋₈ cycloalkyl or 4 to 6-membered heterocycloalkyl        having 1-3 heteroatom ring vertices selected from the group        consisting of O, N, and S;    -   R² and R⁴ are each independently H or C₁₋₃ alkyl;    -   each X¹ is C₁₋₆ alkylene;    -   each R⁵ is independently selected from the group consisting of        hydroxyl, C₃₋₈ cycloalkyl, phenyl, —O-phenyl, —C(O)OR^(a) and        oxo;    -   each R⁶ is C₁₋₈ alkyl or Y, each of which is optionally        substituted with 1-3 substituents selected from the group        consisting of hydroxyl, —O-phenyl, phenyl, and —O—C₁₋₈ alkyl;    -   each R⁷ is independently selected from the group consisting of        C₁₋₈ alkyl, hydroxyl, —O—C₁₋₈ alkyl, oxo, and C(O)OR^(a);    -   each R⁸ is independently selected from the group consisting of        C₁₋₈ alkyl, hydroxyl, and oxo;    -   the subscript n is 0, 1, 2 or 3;    -   each R⁹ is independently selected from the group consisting of        C₁₋₈ alkyl, C₁₋₈ haloalkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl,        —O—X¹—O—C₁₋₈ alkyl, —X¹—O—X¹—O—C₁₋₈ alkyl, —C(O)OR^(a), halogen,        cyano, phenyl, —NR^(b)R^(c), Y, —X¹-C₃₋₈ cycloalkyl, and —X²—Z,        wherein X² is selected from the group consisting of C₁₋₆        alkylene, —C₁₋₆ alkylene-O—, —C₁₋₄ alkylene-O—C₁₋₄ alkylene-,        —C(O)—, and S(O)₂—, Z is 4 to 6-membered heterocycloalkyl having        1-3 heteroatom ring vertices selected from the group consisting        of O, N, and S, and wherein each of said R⁹ substituents is        optionally substituted with 1-3 R¹¹;    -   each of R^(10a), R^(10b), R^(10c) and R^(10d) is independently        selected from the group consisting of C₁₋₈ alkyl, halo, cyano,        —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl, —S(O)₂—C₁₋₆        alkyl, —C(O)NR^(d)R^(e), and 4-6-membered heteroaryl having from        1-3 heteroatom ring vertices selected from the group consisting        of O, N, and S, wherein each of said R^(10a-d) substituents is        optionally substituted with 1-3 R¹², or two of R^(10a), R^(10b),        R^(10c) and R^(10d) on adjacent ring vertices are optionally        combined to form a 5-membered heterocyclic ring optionally        substituted with 1-2 halogens;    -   each R¹¹ is independently selected from the group consisting of        hydroxyl, oxo, halo, cyano, —NR^(d)R^(e), —C(O)OR^(a), phenyl,        C₃₋₈ cycloalkyl, and C₁₋₄ alkyl optionally substituted with        C(O)OR^(a);    -   each R¹² is independently selected from the group consisting of        halo, cyano, hydroxy, —C(O)OR^(a); and    -   each R^(a) is H or C₁₋₆ alkyl;    -   each R^(b) and R^(c) are independently selected from the group        consisting of H, C₁₋₈ alkyl, —S(O)₂—C₁₋₆ alkyl, —C(O)OR^(a), and        —X¹—C(O)OR^(a); and    -   each R^(d) and R^(e) are independently selected from the group        consisting of H, C₁₋₈ alkyl, —S(O)₂—C₁₋₆ alkyl.

In some selected embodiments, the compound of Formula (I) is as providedabove, wherein each R⁹ is independently selected from the groupconsisting of C₁₋₈ alkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈alkyl, —X¹—O—X¹—O—C₁₋₈ alkyl, —C(O)OR^(a), halogen, cyano,

—NR^(b)R^(c), Y, —X¹—C₃₋₈ cycloalkyl, and —X²—Z, wherein X² is selectedfrom the group consisting of C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C₁₋₄alkylene-O—C₁₋₄ alkylene—, —C(O)—, and S(O)₂—, Z is 4 to 6-memberedheterocycloalkyl having 1-3 heteroatom ring vertices selected from thegroup consisting of O, N, and S, and wherein each of said R⁹substituents is optionally substituted with 1-3 R¹¹.

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ia)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ib)

In some selected embodiments, compounds of Formula (I), (Ia), and (Ib)are provided wherein at least one of R^(10a), R^(10b), R^(10c) orR^(10d) is methoxy.

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ic)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Id)

In some selected embodiments, compounds of Formula (I), (Ia), (Ib),(Ic), and (Id) are provided wherein each R⁹ is independently selectedfrom the group consisting of C₁₋₈ alkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈alkyl, —O—X¹—O—C₁₋₈ alkyl, —X¹—O—X¹—O—C₁₋₈ alkyl, wherein each of saidR⁹ substituents is optionally substituted with 1-3 R¹¹.

In some selected embodiments, compounds of Formula (I), (Ia), (Ib),(Ic), and (Id) are provided wherein each R⁹ is independently selectedfrom the group consisting of —C(O)OR^(a), —NR^(b)R^(c), Y, —X¹—C₃₋₈cycloalkyl, and —X²—Z, wherein X² is selected from the group consistingof C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C(O)—, and S(O)₂—, Z is 4 to6-membered heterocycloalkyl having 1-3 heteroatom ring vertices selectedfrom the group consisting of O, N, and S, and wherein each of said R⁹substituents is optionally substituted with 1-3 R¹¹.

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ie)

In some selected embodiments, the compound of Formula (I) is representedby Formula (If)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ig)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ih)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ii)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ij)

In some selected embodiments, the compound of Formula (I) is representedby Formula (Ik)

wherein each R^(x) is independently C₁-C₃ alkyl, and optionally the twoR^(x) moieties are joined together to form a 4, 5, or 6-membered ring.

In some selected embodiments, the compound of Formula (I) is representedby Formula (Il)

wherein each of R^(10a), R^(10b) and R^(10c) is independently selectedfrom the group consisting of H, C₁₋₈ alkyl, halo, cyano, —O—C₁₋₈ alkyl,—X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl, wherein each of said R^(10a),R^(10b) and R^(10c) is optionally substituted with 1-3 R¹².

In some selected embodiments, compounds provided herein are selectedfrom the group consisting of:

In some selected embodiments, any one compound of Table 1 is provided.

Methods of Synthesis

In general, the compounds provided herein can be prepared byconventional methods as described in the Examples below.

Prodrugs and Other Means of Drug Delivery and/or Half-Life Extension

In some aspects of the present invention, compounds described herein areadministered in prodrug form.

In order to effect extension of therapeutic activity, drug molecules maybe engineered to utilize carriers for delivery. Such carriers are eitherused in a non-covalent fashion, with the drug moiety physicochemicallyformulated into a solvent-carrier mixture, or by permanent covalentattachment of a carrier reagent to one of the drug moiety's functionalgroups (see generally WO 20150202317).

Several non-covalent approaches are favored. By way of example, but notlimitation, in certain embodiments depot formulations comprisingnon-covalent drug encapsulation into polymeric carriers are employed. Insuch formulations, the drug molecule is combined with carrier materialand processed such that the drug molecule becomes distributed inside thebulk carrier. Examples include microparticle polymer-drug aggregates(e.g., Degradex® Microspheres (Phosphorex, Inc.)), which areadministered as an injectable suspension; polymer-drug moleculeaggregates formulated as gels (e.g., Lupron Depot® (AbbVie Inc.)), whichare administered as a single bolus injection; and liposomal formulations(e.g., DepoCyt® (Pacira Pharmaceuticals)), where the carrier may be apolymeric or non-polymeric entity capable of solubilizing the drug. Inthese formulations, release of the drug molecule may occur when thecarrier swells or physically deteriorates. In other instances, chemicaldegradation allows diffusion of the drug into the biologicalenvironment; such chemical degradation processes may be autohydrolyticor enzyme-catalyzed. Among other limitations, non-covalent drugencapsulation requires prevention of uncontrolled release of the drug,and dependence of the release mechanism of the drug upon biodegradationmay cause interpatient variability.

In particular embodiments, drug molecules, including both smallmolecules and large molecules, are conjugated to a carrier throughpermanent covalent bonds. Certain small molecule therapeutics thatexhibit low solubility in aqueous fluids may be solubilized byconjugation to hydrophilic polymers, examples of which are describedelsewhere herein. Regarding large molecule proteins, half-life extensionmay be achieved by, for example, permanent covalent modification with apalmitoyl moiety, and by permanent covalent modification with anotherprotein that itself has an extended half-life (e.g., Albuferon®). Ingeneral, drug molecules show decreased biological activity when acarrier is covalently conjugated to the drug.

In certain instances, limitations associated with either drug moleculescomprising non-covalent polymer mixtures or permanent covalentattachment may be successfully addressed by employing a prodrug approachfor chemical conjugation of the drug to the polymer carrier. In thiscontext, therapeutic agents that are inactive or less active than thedrug moiety itself are predictably transformed into active molecularentities. The reduced biological activity of the prodrug as compared tothe released drug is advantageous if a slow or controlled release of thedrug is desired. In such instances, release of the drug occurs overtime, thereby reducing the necessity of repeated and frequentadministration of the drug. A prodrug approach may also be advantageouswhen the drug moiety itself is not absorbed, or has less than optimalabsorption, in the gastrointestinal tract; in these instances, theprodrug facilitates absorption of the drug moiety and is then cleavedoff at some later time (e.g., via first-pass metabolism). Thebiologically active drug molecule is typically linked to the polymericcarrier moiety by a temporary bond formed between the carrier moiety anda hydroxy, amino or carboxy group of the drug molecule.

The approaches described above are associated with several limitations.Prodrug activation may occur by enzymatic or non-enzymatic cleavage ofthe temporary bond between the carrier and the drug molecule, or asequential combination of both (e.g., an enzymatic step followed by anon-enzymatic modification). In an enzyme-free in vitro environment(e.g., an aqueous buffer solution), a temporary bond such as an ester oramide may undergo hydrolysis, but the corresponding rate of hydrolysismay be such that it is outside the therapeutically useful range. Incontrast, in an in vivo environment, esterases or amidases are typicallypresent, and the esterases and amidases may cause significant catalyticacceleration of the kinetics of hydrolysis from two-fold up to severalorders of magnitude (see, e.g., Greenwald et al., (1999) J Med Chem42(18):3857-67).

As described herein, prodrugs may be classified as i) bioprecursors andii) carrier-linked prodrugs. Bioprecursors do not contain a carriergroup and are activated by the metabolic creation of a functional group.In contrast, in carrier-linked prodrugs the active substance isconjugated to a carrier moiety via a temporary linkage at a functionalgroup of the bioactive entity. Preferred functional groups are hydroxylor amino groups. Both the attachment chemistry and hydrolysis conditionsdepend on the type of functional group employed. The carrier may bebiologically inert (e.g., PEG) or may have targeting properties (e.g.,an antibody). Cleavage of the carrier moiety of a carrier-linked prodrugresults in the bioactive entity of interest, and the nature of thedeprotected functional group of the bioactive entity often contributesto its bioactivity.

The patent and scientific literature describe many macromolecularprodrugs where the temporary linkage is a labile ester bond. In thesecases, the functional group of the bioactive entity is either a hydroxylgroup or a carboxylic acid (see, e.g. Cheng et al. (2003) BioconjugateChem 14:1007-17). In addition, it is often advantageous forbiomacromolecules and certain small molecule drugs to link the carrierto an amino group(s) of the bioactive entity (e.g., the N-terminus orlysine amino groups of proteins). During preparation of the prodrug, theamino groups may be more chemoselectively addressed due to their greaternucleophilicity compared to hydroxylic or phenolic groups. This isespecially relevant for proteins and peptides containing a great varietyof different reactive functionalities, where non-selective conjugationreactions lead to undesired product mixtures requiring extensivecharacterization or purification, thus decreasing reaction yield andtherapeutic efficiency of the active moiety.

In general, amide bonds are more stable against hydrolysis than esterbonds, and the rate of cleavage of the amide bond may be too slow fortherapeutic utility in a carrier-linked prodrug. As a result, it may beadvantageous to add structural chemical components in order to effectcontrol over the cleavability of the prodrug amide bond. Theseadditional cleavage-controlling chemical components that are providedneither by the carrier entity nor by the drug are generally referred toas “linkers”. Prodrug linkers can have a major effect on the rate ofhydrolysis of temporary bond, and variation of the chemical nature ofthe linkers often results in particular properties. Prodrug activationof amine-containing biologically active moieties by specific enzymes fortargeted release requires that the structure of the linker display astructural motif recognized as a substrate by a corresponding endogenousenzyme. In these cases, the cleavage of the temporary bond occurs in aone-step process which is catalyzed by the enzyme. For example, theenzymatic release of cytarabin is effected by the protease plasmin,which concentration is relatively high in various kinds of tumor mass.

Interpatient variability is a major drawback of predominant enzymaticcleavage. Enzyme levels may differ significantly between subjectsresulting in biological variation of prodrug activation by the enzymaticcleavage. Enzyme levels may also vary depending on the site ofadministration (e.g., for subcutaneous injection, certain areas of thebody yield more predictable therapeutic effects than others). Inaddition, it is difficult to establish an in vivo—in vitro correlationof the pharmacokinetic properties for enzyme-dependent carrier-linkedprodrugs.

Other carrier prodrugs employing temporary linkages to amino groups inthe drug moiety are based on a cascade mechanism. Cascade cleavage isenabled by linker compounds that are composed of a structuralcombination of a masking group and an activating group. The maskinggroup is attached to the activating group by means of a first temporarylinkage such as an ester or a carbamate. The activating group isattached to an amino group of the drug molecule through a secondtemporary linkage (e.g., a carbamate). The stability or susceptibilityto hydrolysis of the second temporary linkage is dependent on thepresence or absence of the masking group. In the presence of the maskinggroup, the second temporary linkage is highly stable and unlikely torelease the drug molecule with therapeutically useful kinetics, whereasin the absence of the masking group this linkage becomes highly labile,resulting in rapid cleavage and release of the drug moiety.

The cleavage of the first temporary linkage is the rate-limiting step inthe cascade mechanism. The first step may induce a molecularrearrangement of the activating group (e.g., a 1,6-elimination asdescribed in Greenwald et al. (1999) J Med Chem 42:3657-67), and therearrangement renders the second temporary linkage much more labile suchthat its cleavage is induced. Ideally, the cleavage rate of the firsttemporary linkage is identical to the desired release rate for the drugmolecule in a given therapeutic scenario. In addition, it is desirablethat the cleavage of the second temporary linkage be substantiallyinstantaneous after its lability has been induced by cleavage of thefirst temporary bond.

Another embodiment comprises polymeric amino-containing prodrugs basedon trimethyl lock lactonization (see, e.g., Greenwald et al. (2000) JMed Chem 43(3):457-87). In this prodrug system, substitutedo-hydroxyphenyl-dimethylpropionic acid is linked to PEG by an ester,carbonate, or carbamate group as a first temporary linkage and to anamino group of a drug molecule by means of an amide bond as a secondtemporary linkage. The rate-determining step in drug release is theenzymatic cleavage of the first linkage, which is followed by fast amidecleavage by lactonization, releasing an aromatic lactone side product.The primary disadvantage of the prodrug systems described by Greenwaldet al. is the release of highly reactive and potentially toxic aromaticsmall molecule side products like quinone methides or aromatic lactonesafter cleavage of the temporary linkage. The potentially toxic entitiesare released in a 1:1 stoichiometry with the drug and can assume high invivo concentrations.

In certain embodiments of cascade prodrugs comprising aromaticactivating groups based on 1,6-elimination, the masking group isstructurally separate from the carrier. This may be effected byemploying a stable bond between the polymer carrier and the activatinggroup, wherein the stable bond does not participate in the cascadecleavage mechanism. If the carrier is not serving as a masking group andthe activating group is coupled to the carrier by means of a stablebond, release of potentially toxic side products (such as the activatinggroup) is avoided. The stable attachment of the activating group and thepolymer also suppresses the release of drug-linker intermediates withundefined pharmacology.

A first example of the approach described in the preceding paragraphcomprises a polymeric prodrug system based on a mandelic acid activatinggroup (see, e.g., Shabat et al. (2004) Chem Eur J 10:2626-34). In thisapproach the masking group is linked to the activating group by acarbamate bond. The activating group is conjugated permanently to apolyacrylamide polymer via an amide bond. After enzymatic activation ofthe masking group by a catalytic antibody, the masking group is cleavedby cyclization and the drug is released; the activating group is stillconnected to the polyacrylamide polymer after drug release. A similarprodrug system is based on a mandelic acid activating group and anenzymatically cleavable ester-linked masking group (see, e.g., Lee etal. (2004) Angew Chem 116:1707-10).

When the aforementioned linkers are used, the 1,6-elimination step stillgenerates a highly reactive aromatic intermediate. Even if the aromaticmoiety remains permanently attached to the polymeric carrier, sidereactions with potentially toxic by-products or immunogenic effects mayresult. Thus, it is advantageous to generate linker technologies forforming polymeric prodrugs of amine-containing active agents usingaliphatic prodrug linkers that are not enzyme-dependent and do notgenerate reactive aromatic intermediates during cleavage. One suchexample uses PEG5000-maleic anhydride for the reversible modification ofamino groups in tissue-type plasminogen activator and urokinase (see,e.g. (1987) Garman et al. FEBS Lett 223(2):361-65). Regeneration offunctional enzyme from PEG-uPA conjugate upon incubation at pH 7.4buffer by cleavage of the maleamic acid linkage follows first orderkinetics with a half-life of roughly 6 hours. A disadvantage of themaleamic acid linkage is the lack of stability of the conjugate at lowerpH values.

A further approach comprises a PEG cascade prodrug system based onN,N-bis-(2-hydroxyethyl)glycine amide (bicine) linker (see e.g. (2004) JMed Chem 47:726-34). In this system, two PEG carrier molecules arelinked via temporary bonds to a bicine molecule coupled to an aminogroup of the drug molecule. The first steps in prodrug activationinvolves the enzymatic cleavage of the first temporary linkagesconnecting both PEG carrier molecules with the hydroxy groups of thebicine activating group. Different linkages between PEG and bicineresult in different prodrug activation kinetics. The second step inprodrug activation involves the cleavage of the second temporary linkageconnecting the bicine activating group to the amino group of the drugmolecule. A disadvantage of this system is the slow hydrolysis rate ofthis second temporary bicine amide linkage, which results in the releaseof a bicine-modified prodrug intermediate that may show differentpharmacokinetic, immunogenic, toxicity and pharmacodynamic properties ascompared to the native parent drug molecule.

In particular embodiments, dipeptides are utilized for prodrugdevelopment for targeting or targeted transport as they are substratesfor enzymes or biotransport systems. The non-enzymatic route fordipeptide prodrug formation, that is, the ability to undergointramolecular cyclization to form the corresponding diketopiperazine(DKP) and release the active drug, is not well defined.

In some embodiments, dipeptides are attached to a drug moiety via esterbonds, as was described for dipeptide esters of the drug paracetamol(Gomes et al. (2005) Bio & Med Chem Lett). In this case, the cyclizationreaction consists of a nucleophilic attack of the N-terminal amine ofthe peptide on the ester carbon atom to form a tetrahedral intermediate,which is followed by a proton transfer from the amine to the leavinggroup oxyanion with simultaneous formation of a peptide bond to give thecyclic DKP product and free drug. This method is applicable tohydroxyl-containing drugs in vitro but has been found to compete withenzymatic hydrolysis of the ester bond in vivo, as correspondingdipeptide esters released paracetamol at a much faster rate than inbuffer (Gomes et al. (Molecules 12 (2007) 2484-2506). Susceptibility ofdipeptide-based prodrugs to peptidases may be addressed by incorporatingat least one non-natural amino acid in the dipeptide motif. However,endogenous enzymes capable of cleaving ester bonds are not limited topeptidases, and the enzyme-dependence of such prodrug cleavage stillgives rise to unpredictable in vivo performance.

In some embodiments, enzyme-dependence is intentionally engineered intoDKP prodrugs, such as where dipeptide ester prodrugs are formylated atthe amino terminus of the dipeptide, and enzymatic deformylation is usedto initiate diketopiperazine formation and subsequent cleavage of theester-dipeptide bond, followed by release of the drug molecule (see,e.g., U.S. Pat. No. 7,163,923). By way of further example, anoctapeptide is attached by an ester linkage to the 4-hydroxyl group ofvinblastine and undergoes ester bond cleavage by DKP formation afterspecific enzymatic removal of the N-terminal hexapeptide (see Brady etal. (2002) J Med Chem 45:4706-15).

The scope of the DKP formation reaction has also been extended to amideprodrugs. By way of example, U.S. Pat. No. 5,952,294 describes prodrugactivation using diketopiperazine formation for dipeptidyl amideprodrugs of cytarabine. In this case, the temporary linkage is formedbetween the carbonyl of a dipeptide and the aromatic amino group ofcytarabine. However, it is unlikely that a slow-release effect can beachieved for such conjugates as there is no carrier or other half-lifeextending moiety or functionality present.

Dipeptide prodrugs comprising bioactive peptides such as GLP-1 capableof releasing the peptide through diketopiperazine formation of thedipeptidic extension have also been described (see, e.g., WO2009/099763). The bioactive peptide moiety may include an additional PEGchain on one of its amino acid side chain residues to achieve extendedcirculation of the bioactive peptide. However, this approach isassociated with several significant disadvantages. First, the PEG chainhas to be linked to the peptide without compromising its bioactivity,which can be difficult to achieve for many peptide-based bioactiveagents. Second, as the pegylated peptide itself is bioactive, thedipeptidic promoiety has an effect on the peptide's bioactivity and maynegatively affect its receptor binding properties.

Specific exemplary technologies that may be used with the compounds ofthe present invention include those developed by ProLynx (San Francisco,Calif.) and Ascendis Pharma (Palo Alto, Calif.). The ProLynx technologyplatform utilizes sets of novel linkers that are pre-programmed tocleave at different rates to allow the controlled, predictable andsustained release of small molecules and peptides from circulatingsemi-solid macromolecular conjugates. The technology allows formaintenance of desired steady-state serum levels of therapeutic agentsfor weeks to months.

The Ascendis technology platform combines the benefits of prodrug andsustained release technologies to enhance the properties of smallmolecules and peptides. While in circulation, proprietary prodrugsrelease the unmodified active parent therapeutic agent at predeterminedrates governed by physiological pH and temperature conditions. Becausethe therapeutic agent is released in its unmodified form, it retains itsoriginal mechanism of action.

Modifications to Enhance Inhibitor Characteristics

It is frequently beneficial, and sometimes imperative, to improve one ofmore physical properties of the treatment modalities disclosed hereinand/or the manner in which they are administered. Improvements ofphysical properties include, for example, methods of increasing watersolubility, bioavailability, serum half-life, and/or therapeutichalf-life; and/or modulating biological activity.

Modifications known in the art include pegylation, Fc-fusion and albuminfusion. Although generally associated with large molecule agents (e.g.,polypeptides), such modifications have recently been evaluated withparticular small molecules. By way of example, Chiang, M. et al. (J. Am.Chem. Soc., 2014, 136(9):3370-73) describe a small molecule agonist ofthe adenosine 2a receptor conjugated to the immunoglobulin Fc domain.The small molecule-Fc conjugate retained potent Fc receptor andadenosine 2a receptor interactions and showed superior propertiescompared to the unconjugated small molecule. Covalent attachment of PEGmolecules to small molecule therapeutics has also been described (Li, W.et al., Progress in Polymer Science, 2013 38:421-44).

Other known modifications include deuteration to improvepharmacokinetics, pharmacodyanics and toxicity profiles. Due to thegreater atomic mass of deuterium, cleavage of the carbon-deuterium bondrequires more energy than the carbon-hydorgen bond. Because thesestronger bonds are more dfificult to break, the rate of drug metabolismis slower as compared to non-deuterated forms, which allows for lessfrequent dosing and may further reduce toxicities. (Charles Schmidt,Nature Biotechnology, 2017, 35(6): 493-494; Harbeson, S. and Tung, R.,Medchem News, 2014(2): 8-22).

Therapeutic and Prophylactic Uses

The present invention contemplates the use of the A_(2A)R/A_(2B)Rinhibitors described herein in the treatment or prevention of a broadrange of diseases, disorders and/or conditions, and/or the symptomsthereof. While particular uses are described in detail hereafter, it isto be understood that the present invention is not so limited.Furthermore, although general categories of particular diseases,disorders and conditions are set forth hereafter, some of the diseases,disorders and conditions may be a member of more than one category, andothers may not be a member of any of the disclosed categories.

In some embodiments, the diseases, disorders and/or conditions describedherein are mediated, at least in part, by the adenosine A_(2A) receptor(A_(2A)R). In some embodiments, the diseases, disorders and/orconditions described herein are mediated, at least in part, by theadenosine A_(2B) receptor (A_(2B)R). In some embodiments, the diseases,disorders and/or conditions described herein are mediated, at least inpart, by both A_(2A)R and A_(2B)R.

In some embodiments, the A_(2A)R/A_(2B)R inhibitors described herein areadministered in an amount effective to reverse or stop the progressionof A_(2A)R-mediated immunosuppression.

Oncology-related Disorders. As indicated elsewhere herein, in additionto its involvement in the generation of an immune-tolerantmicroenvironment suitable for tumor onset and progression, adenosine,through the engagement of receptors expressed on neoplastic cells, alsoregulates the growth and dissemination of the tumor mass by directactions on cancer cell proliferation, apoptosis and metastasis.Adenosine can also promote cell proliferation via activation of theA_(2A) and A_(2B) receptors.

The pharmacological blockade of A_(2A) receptors results in decreasedcancer development and spread, through an enhancement of the antitumoractions of CD8⁺ T cells as well as via an inhibition of tumorneovascularization, growth and metastatic potential. Likewise, thepharmacological blockade of A_(2B) receptors results in a delay of tumorgrowth and reduction of metastatic dissemination. See, e.g., Antonioli,L. et al., Expert Op on Ther Targets 18(9):973-77 (2014).

In accordance with the present invention, an A_(2A)R/A_(2B)R inhibitorcan be used to treat or prevent a proliferative condition or disorder,including a cancer, for example, cancer of the uterus, cervix, breast,prostate, testes, gastrointestinal tract (e.g., esophagus, oropharynx,stomach, small or large intestines, colon, or rectum), kidney, renalcell, bladder, bone, bone marrow, skin, head or neck, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid,brain (e.g., gliomas), ganglia, central nervous system (CNS) andperipheral nervous system (PNS), and cancers of the hematopoietic systemand the immune system (e.g., spleen or thymus). The present inventionalso provides methods of treating or preventing other cancer-relateddiseases, disorders or conditions, including, for example, immunogenictumors, non-immunogenic tumors, dormant tumors, virus-induced cancers(e.g., epithelial cell cancers, endothelial cell cancers, squamous cellcarcinomas and papillomavirus), adenocarcinomas, lymphomas, carcinomas,melanomas, leukemias, myelomas, sarcomas, teratocarcinomas,chemically-induced cancers, metastasis, and angiogenesis. The inventioncontemplates reducing tolerance to a tumor cell or cancer cell antigen,e.g., by modulating activity of a regulatory T-cell and/or a CD8+ T-cell(see, e.g., Ramirez-Montagut, et al. (2003) Oncogene 22:3180-87; andSawaya, et al. (2003) New Engl. J. Med. 349:1501-09). In particularembodiments, the tumor or cancer is colon cancer, ovarian cancer, breastcancer, melanoma, lung cancer, glioblastoma, or leukemia. The use of theterm(s) cancer-related diseases, disorders and conditions is meant torefer broadly to conditions that are associated, directly or indirectly,with cancer, and includes, e.g., angiogenesis and precancerousconditions such as dysplasia.

In certain embodiments, a cancer be metastatic or at risk of becomingmetastatic, or may occur in a diffuse tissue, including cancers of theblood or bone marrow (e.g., leukemia). In some further embodiments, thecompounds of the invention can be used to overcome T-cell tolerance.

In some embodiments, the present invention provides methods for treatinga proliferative condition, cancer, tumor, or precancerous condition withan A_(2A)R/A_(2B)R inhibitor and at least one additional therapeutic ordiagnostic agent, examples of which are set forth elsewhere herein.

Immune-and Inflammatory-related Disorders. As used herein, terms such as“immune disease”, “immune condition”, “immune disorder”, “inflammatorydisease”, “inflammatory condition”, “inflammatory disorder” and the likeare meant to broadly encompass any immune-related condition (e.g., anautoimmune disease) or a disorder with an inflammatory component thatcan be treated by the A_(2A)R/A_(2B)R inhibitors described herein suchthat some therapeutic benefit is obtained. Such conditions frequentlyare inextricably intertwined with other diseases, disorders andconditions. By way of example, an “immune condition” may refer toproliferative conditions, such as cancer, tumors, and angiogenesis;including infections (acute and chronic), tumors, and cancers thatresist eradication by the immune system.

The A_(2A)R/A_(2B)R inhibitors of the present invention can be used toincrease or enhance an immune response; to improve immunization,including increasing vaccine efficacy; and to increase inflammation.Immune deficiencies associated with immune deficiency diseases,immunosuppressive medical treatment, acute and/or chronic infection, andaging can be treated using the compounds disclosed herein. TheA_(2A)R/A_(2B)R inhibitors can also be used to stimulate the immunesystem of patients suffering from iatrogenically-induced immunesuppression, including those who have undergone bone marrow transplants,chemotherapy, or radiotherapy.

In particular embodiments of the present disclosure, the A_(2A)R/A_(2B)Rinhibitors are used to increase or enhance an immune response to anantigen by providing adjuvant activity. In a particular embodiment, atleast one antigen or vaccine is administered to a subject in combinationwith at least one A_(2A)R/A_(2B)R inhibitor of the present invention toprolong an immune response to the antigen or vaccine. Therapeuticcompositions are also provided which include at least one antigenicagent or vaccine component, including, but not limited to, viruses,bacteria, and fungi, or portions thereof, proteins, peptides,tumor-specific antigens, and nucleic acid vaccines, in combination withat least one A_(2A)R/A_(2B)R inhibitor of the present invention.

A non-limiting list of immune- and inflammatory-related diseases,disorders and conditions which may be treated or prevented with thecompounds and compositions of the present invention include, arthritis(e.g., rheumatoid arthritis), kidney failure, lupus, asthma, psoriasis,colitis, pancreatitis, allergies, fibrosis, surgical complications(e.g., where inflammatory cytokines prevent healing), anemia, andfibromyalgia. Other diseases and disorders which may be associated withchronic inflammation include Alzheimer's disease, congestive heartfailure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis,Parkinson's disease, infections, inflammatory bowel disease (e.g.,Crohn's disease and ulcerative colitis), allergic contact dermatitis andother eczemas, systemic sclerosis, transplantation and multiplesclerosis.

Among other immune-related disorders, it is contemplated that inhibitionof A_(2A)R/A_(2B)R function may also play a role in immunologictolerance and prevention of fetal rejection in utero.

In some embodiments, an A_(2A)R/A_(2B)R inhibitor described herein canbe combined with an immunosuppressive agent to reduce the number ofimmune effector cells.

Some of the aforementioned diseases, disorders and conditions for whichan A_(2A)R/A_(2B)R inhibitor may be particularly efficacious (due to,for example, limitations of current therapies) are described in moredetail hereafter.

Rheumatoid Arthritis (RA), which is generally characterized by chronicinflammation in the membrane lining (the synovium) of the joints,affects approximately 1% of the U.S. population (−2.1 million people).Further understanding of the role of cytokines, including TNF-a andIL-1, in the inflammatory process has enabled the development andintroduction of a new class of disease-modifying antirheumatic drugs(DMARDs). Agents (some of which overlap with treatment modalities forRA) include ENBREL (etanercept), REMICADE (infliximab), HUMIRA(adalimumab) and KINERET (anakinra) Though some of these agents relievesymptoms, inhibit progression of structural damage, and improve physicalfunction in particular patient populations, there is still a need foralternative agents with improved efficacy, complementary mechanisms ofaction, and fewer/less severe adverse effects.

Psoriasis, a constellation of common immune-mediated chronic skindiseases, affects more than 4.5 million people in the U.S., of which 1.5million are considered to have a moderate-to severe form of the disease.Moreover, over 10% of patients with psoriasis develop psoriaticarthritis, which damages the bone and connective tissue around thejoints. An improved understanding of the underlying physiology ofpsoriasis has resulted in the introduction of agents that, for example,target the activity of T lymphocytes and cytokines responsible for theinflammatory nature of the disease. Such agents include the TNF-αinhibitors (also used in the treatment of rheumatoid arthritis (RA)),including ENBREL (etanercept), REMICADE (infliximab) and HUMIRA(adalimumab)), and T-cell inhibitors such as AMEVIVE (alefacept) andRAPTIVA (efalizumab). Though several of these agents are effective tosome extent in certain patient populations, none have been shown toeffectively treat all patients.

Microbial-related Disorders. The present invention contemplates the useof the A_(2A)R/A_(2B)R inhibitors described herein in the treatmentand/or prevention of any viral, bacterial, fungal, parasitic or otherinfective disease, disorder or condition for which treatment with anA_(2A)R/A_(2B)R inhibitor may be beneficial.

Examples of viral diseases, disorders and conditions that arecontemplated include, but are not limited to, hepatitis B virus (HBV),hepatitis C virus (HCV), human papilloma virus (HPV), HIV, AIDS(including its manifestations such as cachexia, dementia, and diarrhea),herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella zostervirus, coxsackie virus, and cytomegalovirus (CMV).

Further examples of such diseases and disorders include staphylococcaland streptococcal infections (e.g., Staphylococcus aureus andstreptococcus sanguinis, respectively), leishmania, toxoplasma,trichomonas, giardia, candida albicans, bacillus anthracis, andpseudomonas aeruginosa. In some embodiments, diseases or disordersinclude Mycobacterium infection (e.g., Mycobacterium leprae orMycobacterium tuberculosis) or an infection caused by Listeriamonocytogenes or Toxplasma gondii. Compounds of the invention can beused to treat sepsis, decrease or inhibit bacterial growth, and reduceor inhibit inflammatory cytokines.

Further embodiments contemplate the treatment of a parasitic infectionincluding, but not limited to, Leishmania donovani, Leishmania tropica,Leishmania major, Leishmania aethiopica, Leishmania mexicana, Plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.Frequently, anti-parasitic therapy is administered prophylactically(e.g., before a subject travels to an area with a high frequency ofparasitic infection).

CNS-related and Neurological Disorders. Inhibition of A_(2A)R/A_(2B)Rmay also be an important treatment strategy for patients withneurological, neuropsychiatric, neurodegenerative or other diseases,disorders and conditions having some association with the centralnervous system, including disorders associated with impairment ofcognitive function and motor function. Examples include Parkinson'sdisease, extra pyramidal syndrome (EPS), dystonia, akathisia, tardivedyskinesia, restless leg syndrome (RLS), epilepsy, periodic limbmovement in sleep (PLMS), attention deficit disorders, depression,anxiety, dementia, Alzheimer's disease, Huntington's disease, multiplesclerosis, cerebral ischemia, hemorrhagic stroke, subarachnoidhemorrhage, and traumatic brain injury.

Subjects suffering from multiple sclerosis (MS), a seriouslydebilitating autoimmune disease comprising multiple areas ofinflammation and scarring of the myelin in the brain and spinal cord,may be particularly helped by the A_(2A)R/A_(2B)R inhibitors describedherein, as current treatments only alleviate symptoms or delay theprogression of disability.

Similarly, the A_(2A)R/A_(2B)R inhibitors may be particularlyadvantageous for subjects afflicted with neurodegenerative disorders,such as Alzheimer's disease (AD), a brain disorder that seriouslyimpairs patients' thought, memory, and language processes; andParkinson's disease (PD), a progressive disorder of the CNScharacterized by, for example, abnormal movement, rigidity and tremor.These disorders are progressive and debilitating, and no curative agentsare available.

Other Disorders. Embodiments of the present invention contemplate theadministration of the A_(2A)R/A_(2B)R inhibitors described herein to asubject for the treatment or prevention of any other disorder that maybenefit from at least some level of A_(2A)R/A_(2B)R inhibition. Suchdiseases, disorders and conditions include, for example, cardiovascular(e.g., cardiac ischemia), gastrointestinal (e.g., Crohn's disease),metabolic (e.g., diabetes), hepatic (e.g., hepatic fibrosis, NASH, andNAFLD), pulmonary (e.g., COPD and asthma), ophthalmologic (e.g.,diabetic retinopathy), and renal (e.g., renal failure) disorders.

Pharmaceutical Compositions

The A_(2A)R/A_(2B)R inhibitors of the present invention may be in theform of compositions suitable for administration to a subject. Ingeneral, such compositions are “pharmaceutical compositions” comprisingan A_(2A)R/A_(2B)R inhibitor(s) and one or more pharmaceuticallyacceptable or physiologically acceptable diluents, carriers orexcipients. In certain embodiments, the A_(2A)R/A_(2B)R inhibitors arepresent in a therapeutically acceptable amount. The pharmaceuticalcompositions may be used in the methods of the present invention; thus,for example, the pharmaceutical compositions can be administered ex vivoor in vivo to a subject in order to practice the therapeutic andprophylactic methods and uses described herein.

The pharmaceutical compositions of the present invention can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds as describedherein in order to treat or prevent the diseases, disorders andconditions as contemplated by the present invention.

The pharmaceutical compositions containing the active ingredient (e.g.,an inhibitor of A_(2A)R/A_(2B)R function) may be in a form suitable fororal use, for example, as tablets, capsules, troches, lozenges, aqueousor oily suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, or syrups, solutions, microbeads or elixirs.Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions, and such compositions may contain one ormore agents such as, for example, sweetening agents, flavoring agents,coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets, capsulesand the like contain the active ingredient in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be, for example, diluents,such as calcium carbonate, sodium carbonate, lactose, calcium phosphateor sodium phosphate; granulating and disintegrating agents, for example,corn starch, or alginic acid; binding agents, for example starch,gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylene-vinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present invention may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally occurring gums, for example, gum acacia or gumtragacanth; naturally occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of an A_(2A)R/A_(2B)R inhibitor contemplated by thepresent invention and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, detergents,buffers, vehicles, diluents, and/or adjuvants. For example, a suitablevehicle may be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers that canbe used in the pharmaceutical compositions and dosage forms contemplatedherein. Typical buffers include, but are not limited to,pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.As an example, the buffer components can be water soluble materials suchas phosphoric acid, tartaric acids, lactic acid, succinic acid, citricacid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, andsalts thereof. Acceptable buffering agents include, for example, a Trisbuffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including liposomes, hydrogels, prodrugsand microencapsulated delivery systems. For example, a time delaymaterial such as glyceryl monostearate or glyceryl stearate alone, or incombination with a wax, may be employed. Any drug delivery apparatus maybe used to deliver an A_(2A)R/A_(2B)R inhibitor, including implants(e.g., implantable pumps) and catheter systems, slow injection pumps anddevices, all of which are well known to the skilled artisan.

Depot injections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the A_(2A)R/A_(2B)Rinhibitors disclosed herein over a defined period of time. Depotinjections are usually either solid- or oil-based and generally compriseat least one of the formulation components set forth herein. One ofordinary skill in the art is familiar with possible formulations anduses of depot injections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. Moreover, fatty acids such as oleic acid, find use inthe preparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The present invention contemplates the administration of theA_(2A)R/A_(2B)R inhibitors in the form of suppositories for rectaladministration. The suppositories can be prepared by mixing the drugwith a suitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials include, but arenot limited to, cocoa butter and polyethylene glycols.

The A_(2A)R/A_(2B)R inhibitors contemplated by the present invention maybe in the form of any other suitable pharmaceutical composition (e.g.,sprays for nasal or inhalation use) currently known or developed in thefuture.

Routes of Administration

The present invention contemplates the administration of A_(2A)R/A_(2B)Rinhibitors, and compositions thereof, in any appropriate manner.Suitable routes of administration include oral, parenteral (e.g.,intramuscular, intravenous, subcutaneous (e.g., injection or implant),intraperitoneal, intracisternal, intraarticular, intraperitoneal,intracerebral (intraparenchymal) and intracerebroventricular), nasal,vaginal, sublingual, intraocular, rectal, topical (e.g., transdermal),buccal and inhalation. Depot injections, which are generallyadministered subcutaneously or intramuscularly, may also be utilized torelease the A_(2A)R/A_(2B)R inhibitors disclosed herein over a definedperiod of time.

Particular embodiments of the present invention contemplate oraladministration.

Combination Therapy

The present invention contemplates the use of A_(2A)R/A_(2B)R inhibitorsalone or in combination with one or more active therapeutic agents. Theadditional active therapeutic agents can be small chemical molecules;macromolecules such as proteins, antibodies, peptibodies, peptides, DNA,RNA or fragments of such macromolecules; or cellular or gene therapies.In such combination therapy, the various active agents frequently havedifferent, complementary mechanisms of action. Such combination therapymay be especially advantageous by allowing a dose reduction of one ormore of the agents, thereby reducing or eliminating the adverse effectsassociated with one or more of the agents. Furthermore, such combinationtherapy may have a synergistic therapeutic or prophylactic effect on theunderlying disease, disorder, or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, the A_(2A)R/A_(2B)R inhibitors are administeredor applied sequentially, e.g., where one agent is administered prior toone or more other agents. In other embodiments, the A_(2A)R/A_(2B)Rinhibitors are administered simultaneously, e.g., where two or moreagents are administered at or about the same time; the two or moreagents may be present in two or more separate formulations or combinedinto a single formulation (i.e., a co-formulation). Regardless ofwhether the two or more agents are administered sequentially orsimultaneously, they are considered to be administered in combinationfor purposes of the present invention.

The A_(2A)R/A_(2B)R inhibitors of the present invention may be used incombination with at least one other (active) agent in any mannerappropriate under the circumstances. In one embodiment, treatment withthe at least one active agent and at least one A_(2A)R/A_(2B)R inhibitorof the present invention is maintained over a period of time. In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment with anA_(2A)R/A_(2B)R inhibitor of the present invention is maintained at aconstant dosing regimen. In a further embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with an A_(2A)R/A_(2B)R inhibitor ofthe present invention is reduced (e.g., lower dose, less frequent dosingor shorter treatment regimen). In yet another embodiment, treatment withthe at least one active agent is reduced or discontinued (e.g., when thesubject is stable), and treatment with the A_(2A)R/A_(2B)R inhibitor ofthe present invention is increased (e.g., higher dose, more frequentdosing or longer treatment regimen). In yet another embodiment,treatment with the at least one active agent is maintained and treatmentwith the A_(2A)R/A_(2B)R inhibitor of the present invention is reducedor discontinued (e.g., lower dose, less frequent dosing or shortertreatment regimen). In yet another embodiment, treatment with the atleast one active agent and treatment with the A_(2A)R/A_(2B)R inhibitorof the present invention are reduced or discontinued (e.g., lower dose,less frequent dosing or shorter treatment regimen).

Oncology-related Disorders. The present invention provides methods fortreating and/or preventing a proliferative condition, cancer, tumor, orprecancerous disease, disorder or condition with an A_(2A)R/A_(2B)Rinhibitor and at least one additional therapeutic or diagnostic agent.In some embodiments, the additional therapeutic or diagnostic agent isradiation, an immunomodulatory agent or chemotherapeutic agent, ordiagnostic agent. Suitable immunomodulatory agents that may be used inthe present invention include CD4OL, B7, and B7RP1; activatingmonoclonal antibodies (mAbs) to stimulatory receptors, such as,anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligand; dendritic cellantigen loading (in vitro or in vivo); anti-cancer vaccines such asdendritic cell cancer vaccines; cytokines/chemokines, such as, ILL IL2,IL12, IL18, ELC/CCL19, SLC/CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC,IFNa/b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacteriallipopolysaccharides (LPS); indoleamine 2,3-dioxygenase 1 (IDO1)inhibitors and immune-stimulatory oligonucleotides.

In certain embodiments, the present invention provides methods for tumorsuppression of tumor growth comprising administration of anA_(2A)R/A_(2B)R inhibitor described herein in combination with a signaltransduction inhibitor (STI) to achieve additive or synergisticsuppression of tumor growth. As used herein, the term “signaltransduction inhibitor” refers to an agent that selectively inhibits oneor more steps in a signaling pathway. Signal transduction inhibitors(STIs) of the present invention include: (i) bcr/abl kinase inhibitors(e.g., GLEEVEC); (ii) epidermal growth factor (EGF) receptor inhibitors,including kinase inhibitors and antibodies; (iii) her-2/neu receptorinhibitors (e.g., HERCEPTIN); (iv) inhibitors of Akt family kinases orthe Akt pathway (e.g., rapamycin); (v) cell cycle kinase inhibitors(e.g., flavopiridol); and (vi) phosphatidyl inositol kinase inhibitors.Agents involved in in immunomodulation can also be used in combinationwith the A_(2A)R/A_(2B)R inhibitors described herein for the suppressionof tumor growth in cancer patients.

Examples of chemotherapeutic agents include, but are not limited to,alkylating agents such as thiotepa and cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethylenethiophosphaoramide andtrimethylolomelamime; nitrogen mustards such as chiorambucil,chlornaphazine, cholophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, calicheamicin,carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin,epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin,puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin,tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such asmethotrexate and 5-fluorouracil (5-FU); folic acid analogs such asdenopterin, methotrexate, pteropterin, trimetrexate; purine analogs suchas fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine;mercaptopurine; methotrexate; platinum and platinum coordinationcomplexes such as cisplatin, carboplatin and oxaliplatin; vinblastine;etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine;vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin;xeloda; ibandronate; CPT11; topoisomerase inhibitors;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; anthracyclines; and pharmaceutically acceptable salts,acids or derivatives of any of the above.

Chemotherapeutic agents also include anti-hormonal agents that act toregulate or inhibit hormonal action on tumors such as anti-estrogens,including for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, onapristone,and toremifene; and antiandrogens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptablesalts, acids or derivatives of any of the above. In certain embodiments,combination therapy comprises a chemotherapy regimen that includes oneor more chemotherapeutic agents. In certain embodiments, combinationtherapy comprises administration of a hormone or related hormonal agent.

Additional treatment modalities that may be used in combination with anA_(2A)R/A_(2B)R inhibitor include radiotherapy, a monoclonal antibodyagainst a tumor antigen, a complex of a monoclonal antibody and toxin, aT-cell adjuvant, bone marrow transplant, or antigen presenting cells(e.g., dendritic cell therapy), including TLR agonists which are used tostimulate such antigen presenting cells.

In certain embodiments, the present invention contemplates the use ofthe compounds described herein in combination with adoptive celltherapy, a new and promising form of personalized immunotherapy in whichimmune cells with anti-tumor activity are administered to cancerpatients. Adoptive cell therapy is being explored usingtumor-infiltrating lymphocytes (TIL) and T cells engineered to express,for example, chimeric antigen receptors (CAR) or T cell receptors (TCR).Adoptive cell therapy generally involves collecting T cells from anindividual, genetically modifying them to target a specific antigen orto enhance their anti-tumor effects, amplifying them to a sufficientnumber, and infusion of the genetically modified T cells into a cancerpatient. T cells can be collected from the patient to whom the expandedcells are later reinfused (e.g., autologous) or can be collected fromdonor patients (e.g., allogeneic).

In certain embodiments, the present invention contemplates the use ofthe compounds described herein in combination with RNAinterference-based therapies to silence gene expression. RNAi beginswith the cleavage of longer double-stranded RNAs into small interferingRNAs (siRNAs). One strand of the siRNA is incorporated into aribonucleoprotein complex known as the RNA-induced silencing complex(RISC), which is then used to identify mRNA molecules that are at leastpartially complementary to the incorporated siRNA strand. RISC can bindto or cleave the mRNA, both of which inhibits translation.

Immune Checkpoint Inhibitors. The present invention contemplates the useof the inhibitors of A_(2A)R/A_(2B)R function described herein incombination with immune checkpoint inhibitors.

The tremendous number of genetic and epigenetic alterations that arecharacteristic of all cancers provides a diverse set of antigens thatthe immune system can use to distinguish tumor cells from their normalcounterparts. In the case of T cells, the ultimate amplitude (e.g.,levels of cytokine production or proliferation) and quality (e.g., thetype of immune response generated, such as the pattern of cytokineproduction) of the response, which is initiated through antigenrecognition by the T-cell receptor (TCR), is regulated by a balancebetween co-stimulatory and inhibitory signals (immune checkpoints).Under normal physiological conditions, immune checkpoints are crucialfor the prevention of autoimmunity (i.e., the maintenance ofself-tolerance) and also for the protection of tissues from damage whenthe immune system is responding to pathogenic infection. The expressionof immune checkpoint proteins can be dysregulated by tumors as animportant immune resistance mechanism.

T-cells have been the major focus of efforts to therapeuticallymanipulate endogenous antitumor immunity because of i) their capacityfor the selective recognition of peptides derived from proteins in allcellular compartments; ii) their capacity to directly recognize and killantigen-expressing cells (by CD8+ effector T cells; also known ascytotoxic T lymphocytes (CTLs)); and iii) their ability to orchestratediverse immune responses by CD4+ helper T cells, which integrateadaptive and innate effector mechanisms.

In the clinical setting, the blockade of immune checkpoints—whichresults in the amplification of antigen-specific T cell responses—hasshown to be a promising approach in human cancer therapeutics.

T cell-mediated immunity includes multiple sequential steps, each ofwhich is regulated by counterbalancing stimulatory and inhibitorysignals in order to optimize the response. While nearly all inhibitorysignals in the immune response ultimately modulate intracellularsignaling pathways, many are initiated through membrane receptors, theligands of which are either membrane-bound or soluble (cytokines). Whileco-stimulatory and inhibitory receptors and ligands that regulate T-cellactivation are frequently not over-expressed in cancers relative tonormal tissues, inhibitory ligands and receptors that regulate T celleffector functions in tissues are commonly overexpressed on tumor cellsor on non-transformed cells associated with the tumor microenvironment.The functions of the soluble and membrane-bound receptor—ligand immunecheckpoints can be modulated using agonist antibodies (forco-stimulatory pathways) or antagonist antibodies (for inhibitorypathways). Thus, in contrast to most antibodies currently approved forcancer therapy, antibodies that block immune checkpoints do not targettumor cells directly, but rather target lymphocyte receptors or theirligands in order to enhance endogenous antitumor activity. [See Pardoll,(April 2012) Nature Rev. Cancer 12:252-64].

Examples of immune checkpoints (ligands and receptors), some of whichare selectively upregulated in various types of tumor cells, that arecandidates for blockade include PD1 (programmed cell death protein 1);PDL1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4(cytotoxic T-lymphocyte associated antigen 4); TIM3 (T-cell membraneprotein 3); LAG3 (lymphocyte activation gene 3); TIGIT (T cellimmunoreceptor with Ig and ITIM domains); and Killer InhibitoryReceptors, which can be divided into two classes based on theirstructural features: i) killer cell immunoglobulin-like receptors(KIRs), and ii) C-type lectin receptors (members of the type IItransmembrane receptor family). Other less well-defined immunecheckpoints have been described in the literature, including bothreceptors (e.g., the 2B4 (also known as CD244) receptor) and ligands(e.g., certain B7 family inhibitory ligands such B7-H3 (also known asCD276) and B7-H4 (also known as B7-S1, B7x and VCTN1)). [See Pardoll,(April 2012) Nature Rev. Cancer 12:252-64].

The present invention contemplates the use of the inhibitors ofA_(2A)R/A_(2B)R function described herein in combination with inhibitorsof the aforementioned immune-checkpoint receptors and ligands, as wellas yet-to-be-described immune-checkpoint receptors and ligands. Certainmodulators of immune checkpoints are currently approved, and many othersare in development. When it was approved for the treatment of melanomain 2011, the fully humanized CTLA4 monoclonal antibody ipilimumab(YERVOY; Bristol-Myers Squibb) became the first immune checkpointinhibitor to receive regulatory approval in the US. Fusion proteinscomprising CTLA4 and an antibody (CTLA4-Ig; abatcept (ORENCIA;Bristol-Myers Squibb)) have been used for the treatment of rheumatoidarthritis, and other fusion proteins have been shown to be effective inrenal transplantation patients that are sensitized to Epstein BarrVirus. The next class of immune checkpoint inhibitors to receiveregulatory approval were against PD-1 and its ligands PD-L1 and PD-L2.Approved anti-PD1 antibodies include nivolumab (OPDIVO; Bristol-MyersSquibb) and pembrolizumab (KEYTRUDA; Merck) for various cancers,including squamous cell carcinoma, classical Hodgkin lymphoma andurothelial carcinoma. Approved anti-PDL1 antibodies include avelumab(BAVENCIO, EMD Serono & Pfizer), atezolizumab (TECENTRIQ;Roche/Genentech), and durvalumab (IMFINZI; AstraZeneca) for certaincancers, including urothelial carcinoma. While there are no approvedtherapeutics targeting TIGIT or its ligands CD155 and CD112, those indevelopment include BMS-986207 (Bristol-Myers Squibb), MTIG7192A/RG6058(Roche/Genentech), and OMP-31M32 (OncoMed).

In one aspect of the present invention, the claimed A_(2A)R/A_(2B)Rinhibitors are combined with an immuno-oncology agent that is (i) anagonist of a stimulatory (including a co-stimulatory) receptor or (ii)an antagonist of an inhibitory (including a co-inhibitory) signal on Tcells, both of which result in amplifying antigen-specific T cellresponses. Certain of the stimulatory and inhibitory molecules aremembers of the immunoglobulin super family (IgSF). One important familyof membrane-bound ligands that bind to co-stimulatory or co-inhibitoryreceptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1),B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), B7-H6, andB7-H7 (HHLA2). Another family of membrane bound ligands that bind toco-stimulatory or co-inhibitory receptors is the TNF family of moleculesthat bind to cognate TNF receptor family members, which includes CD40and CD4OL, OX-40, OX-40L, CD70, CD27L, CD30, CD3OL, 4-1BBL, CD137(4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4, OPG,RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA,LT13R, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3, EDAR, EDA1, XEDAR, EDA2,TNFR1, Lymphotoxin a/TNF13, TNFR2, TNFa, LT13R, Lymphotoxin a 1132, FAS,FASL, RELT, DR6, TROY, NGFR.

In another aspect, the immuno-oncology agent is a cytokine that inhibitsT cell activation (e.g., IL-6, IL-10, TGF-B, VEGF, and otherimmunosuppressive cytokines) or a cytokine that stimulates T cellactivation, for stimulating an immune response.

In one aspect, T cell responses can be stimulated by a combination ofthe disclosed A_(2A)R/A_(2B)R inhibitors and one or more of (i) anantagonist of a protein that inhibits T cell activation (e.g., immunecheckpoint inhibitors) such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3,Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56,VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and/or (ii) anagonist of a protein that stimulates T cell activation such as B7-1,B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX4OL, GITR,GITRL, CD70, CD27, CD40, DR3 and CD2. Other agents that can be combinedwith the A_(2A)R/A_(2B)R inhibitors of the present invention for thetreatment of cancer include antagonists of inhibitory receptors on NKcells or agonists of activating receptors on NK cells. For example,compounds herein can be combined with antagonists of KIR, such aslirilumab.

Yet other agents for combination therapies include agents that inhibitor deplete macrophages or monocytes, including but not limited to CSF-1Rantagonists such as CSF-1R antagonist antibodies including RG7155(W011/70024, W011/107553, W011/131407, W013/87699, W013/119716,W013/132044) or FPA-008 (W011/140249; W013169264; W014/036357).

In another aspect, the disclosed A_(2A)R/A_(2B)R inhibitors can be usedwith one or more of agonistic agents that ligate positive costimulatoryreceptors, blocking agents that attenuate signaling through inhibitoryreceptors, antagonists, and one or more agents that increasesystemically the frequency of anti-tumor T cells, agents that overcomedistinct immune suppressive pathways within the tumor microenvironment(e.g., block inhibitory receptor engagement (e.g., PD-L1/PD-1interactions), deplete or inhibit Tregs (e.g., using an anti-CD25monoclonal antibody (e.g., daclizumab) or by ex vivo anti-CD25 beaddepletion), or reverse/prevent T cell anergy or exhaustion) and agentsthat trigger innate immune activation and/or inflammation at tumorsites.

In one aspect, the immuno-oncology agent is a CTLA-4 antagonist, such asan antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, forexample, YERVOY (ipilimumab) or tremelimumab.

In another aspect, the immuno-oncology agent is a PD-1 antagonist, suchas an antagonistic PD-1 antibody. Suitable PD-1 antibodies include, forexample, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680(AMP-514; W02012/145493). The immuno-oncology agent may also includepidilizumab (CT-011), though its specificity for PD-1 binding has beenquestioned. Another approach to target the PD-1 receptor is therecombinant protein composed of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgGl, called AMP-224.

In another aspect, the immuno-oncology agent is a PD-L1 antagonist, suchas an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include,for example, TECENTRIC (atezolizumab; MPDL3280A; WO2010/077634),durvalumab (MEDI4736), BMS-936559 (W02007/005874), and MSB0010718C(W02013/79174).

In another aspect, the immuno-oncology agent is a LAG-3 antagonist, suchas an antagonistic LAG-3 antibody. Suitable LAG3 antibodies include, forexample, BMS-986016 (W010/19570, W014/08218), or IMP-731 or IMP-321(W008/132601, W009/44273).

In another aspect, the immuno-oncology agent is a CD137 (4-1BB) agonist,such as an agonistic CD137 antibody. Suitable CD137 antibodies include,for example, urelumab and PF-05082566 (W012/32433).

In another aspect, the immuno-oncology agent is a GITR agonist, such asan agonistic GITR antibody. Suitable GITR antibodies include, forexample, BMS-986153, BMS-986156, TRX-518 (W006/105021, W009/009116) andMK-4166 (W011/028683).

In another aspect, the immuno-oncology agent is an OX40 agonist, such asan agonistic OX40 antibody. Suitable OX40 antibodies include, forexample, MEDI-6383 or MEDI-6469.

In another aspect, the immuno-oncology agent is an OX4OL antagonist,such as an antagonistic OX40 antibody. Suitable OX4OL antagonistsinclude, for example, RG-7888 (W006/029879).

In another aspect, the immuno-oncology agent is a CD40 agonist, such asan agonistic CD40 antibody. In yet another embodiment, theimmuno-oncology agent is a CD40 antagonist, such as an antagonistic CD40antibody. Suitable CD40 antibodies include, for example, lucatumumab ordacetuzumab.

In another aspect, the immuno-oncology agent is a CD27 agonist, such asan agonistic CD27 antibody. Suitable CD27 antibodies include, forexample, varlilumab.

In another aspect, the immuno-oncology agent is MGA271 (to B7H3)(W011/109400).

The present invention encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Metabolic and Cardiovascular Diseases. The present invention providesmethods for treating and/or preventing certain cardiovascular- and/ormetabolic-related diseases, disorders and conditions, as well asdisorders associated therewith, with an A_(2A)R/A_(2B)R inhibitor and atleast one additional therapeutic or diagnostic agent.

Examples of therapeutic agents useful in combination therapy for thetreatment of hypercholesterolemia (and atherosclerosis as well) includestatins (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR, PRAVACOL, and ZOCOR),which inhibit the enzymatic synthesis of cholesterol; bile acid resins(e.g., COLESTID, LO-CHOLEST, PREVALITE, QUESTRAN, and WELCHOL), whichsequester cholesterol and prevent its absorption; ezetimibe (ZETIA),which blocks cholesterol absorption; fibric acid (e.g., TRICOR), whichreduces triglycerides and may modestly increase HDL; niacin (e.g.,NIACOR), which modestly lowers LDL cholesterol and triglycerides; and/ora combination of the aforementioned (e.g., VYTORIN (ezetimibe withsimvastatin). Alternative cholesterol treatments that may be candidatesfor use in combination with the A_(2A)R/A_(2B)R inhibitors describedherein include various supplements and herbs (e.g., garlic, policosanol,and guggul).

The present invention encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Immune-and Inflammatory-related Disorders. The present inventionprovides methods for treating and/or preventing immune-related diseases,disorders and conditions; and diseases, disorders and conditions havingan inflammatory component; with an A_(2A)R/A_(2B)R inhibitor and atleast one additional therapeutic or diagnostic agent.

Examples of therapeutic agents useful in combination therapy include,but are not limited to, the following: non-steroidal anti-inflammatorydrug (NSAID) such as aspirin, ibuprofen, and other propionic acidderivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen,fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen,miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen,tiaprofenic acid, and tioxaprofen), acetic acid derivatives(indomethacin, acemetacin, alclofenac, clidanac, diclofenac,fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac,oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac),fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamicacid, niflumic acid and tolfenamic acid), biphenylcarboxylic acidderivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam,sudoxicam and tenoxican), salicylates (acetyl salicylic acid,sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone,mofebutazone, oxyphenbutazone, phenylbutazone). Other combinationsinclude cyclooxygenase-2 (COX-2) inhibitors.

Other active agents for combination include steroids such asprednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, or hydrocortisone. Such a combination may be especiallyadvantageous since one or more adverse effects of the steroid can bereduced or even eliminated by tapering the steroid dose required.

Additional examples of active agents that may be used in combinationsfor treating, for example, rheumatoid arthritis, include cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); antibodies to, orantagonists of, other human cytokines or growth factors, for example,TNF, LT, IL-10, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, or PDGF.

Particular combinations of active agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade, andinclude TNF antagonists such as chimeric, humanized or human TNFantibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870), andsoluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG(ENBREL.) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor (sIL-13),and also TNFa-converting enzyme (TACE) inhibitors; similarly, IL-1inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) may beeffective. Other combinations include Interleukin 11, anti-P7s andp-selectin glycoprotein ligand (PSGL). Other examples of agents usefulin combination with the A_(2A)R/A_(2B)R inhibitors described hereininclude interferon-131a (AVONEX); interferon-131b (BETASERON); copaxone;hyperbaric oxygen; intravenous immunoglobulin; clabribine; andantibodies to, or antagonists of, other human cytokines or growthfactors (e.g., antibodies to CD40 ligand and CD80).

Microbial Diseases. The present invention provides methods for treatingand/or preventing viral, bacterial, fungal and parasitic diseases,disorders and conditions, as well as disorders associated therewith,with an A_(2A)R/A_(2B)R inhibitor and at least one additionaltherapeutic or diagnostic agent (e.g., one or more other antiviralagents and/or one or more agents not associated with viral therapy).

Such combination therapy includes anti-viral agents targeting variousviral life-cycle stages and having different mechanisms of action,including, but not limiting to, the following: inhibitors of viraluncoating (e.g., amantadine and rimantidine); reverse transcriptaseinhibitors (e.g., acyclovir, zidovudine, and lamivudine); agents thattarget integrase; agents that block attachment of transcription factorsto viral DNA; agents (e.g., antisense molecules) that impact translation(e.g., fomivirsen); agents that modulate translation/ribozyme function;protease inhibitors; viral assembly modulators (e.g., rifampicin);antiretrovirals such as, for example, nucleoside analogue reversetranscriptase inhibitors (e.g., azidothymidine (AZT), ddl, ddC, 3TC,d4T); non-nucleoside reverse transcriptase inhibitors (e.g., efavirenz,nevirapine); nucleotide analogue reverse transcriptase inhibitors; andagents that prevent release of viral particles (e.g., zanamivir andoseltamivir). Treatment and/or prevention of certain viral infections(e.g., HIV) frequently entail a group (“cocktail”) of antiviral agents.

Other antiviral agents contemplated for use in combination with anA_(2A)R/A_(2B)R inhibitor include, but are not limited to, thefollowing: abacavir, adefovir, amantadine, amprenavir, ampligen,arbidol, atazanavir, atripla, boceprevirertet, cidofovir, combivir,darunavir, delavirdine, didanosine, docosanol, edoxudine, emtricitabine,enfuvirtide, entecavir, famciclovir, fosamprenavir, foscarnet, fosfonet,http://en.wikipedia.org/wiki/Fusion_inhibitor ganciclovir, ibacitabine,imunovir, idoxuridine, imiquimod, indinavir, inosine, variousinterferons (e.g., peginterferon alfa-2a), lopinavir, loviride,maraviroc, moroxydine, methisazone, nelfinavir, nexavir, penciclovir,peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin,ritonavir, pyramidine, saquinavir, stavudine, telaprevir, tenofovir,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, and zalcitabine.

The present invention contemplates the use of the inhibitors ofA_(2A)R/A_(2B)R function described herein in combination withantiparasitic agents. Such agents include, but are not limited to,thiabendazole, pyrantel pamoate, mebendazole, praziquantel, niclosamide,bithionol, oxamniquine, metrifonate, ivermectin, albendazole,eflornithine, melarsoprol, pentamidine, benznidazole, nifurtimox, andnitroimidazole. The skilled artisan is aware of other agents that mayfind utility for the treatment of parasitic disorders.

Embodiments of the present invention contemplate the use of theA_(2A)R/A_(2B)R inhibitors described herein in combination with agentsuseful in the treatment or prevention of bacterial disorders.Antibacterial agents can be classified in various manners, includingbased on mechanism of action, based on chemical structure, and based onspectrum of activity. Examples of antibacterial agents include thosethat target the bacterial cell wall (e.g., cephalosporins andpenicillins) or the cell membrane (e.g., polymyxins), or interfere withessential bacterial enzymes (e.g., sulfonamides, rifamycins, andquinolines). Most antibacterial agents that target protein synthesis(e.g., tetracyclines and macrolides) are bacteriostatic, whereas agentssuch as the aminoglycoside are bactericidal. Another means ofcategorizing antibacterial agents is based on their target specificity;“narrow-spectrum” agents target specific types of bacteria (e.g.,Gram-positive bacteria such as Streptococcus), while “broad-spectrum”agents have activity against a broader range of bacteria. The skilledartisan is aware of types of anti-bacterial agents that are appropriatefor use in specific bacterial infections.

Embodiments of the present invention contemplate the use of theA_(2A)R/A_(2B)R inhibitors described herein in combination with agentsuseful in the treatment or prevention of fungal disorders. Antifungalagents include polyenes (e.g., amphotericin, nystatin, and pimaricin);azoles (e.g., fluconazole, itraconazole, and ketoconazole); allylamines(e.g., naftifine, and terbinafine) and morpholines (e.g., amorolfine);and antimetabolies (e.g., 5-fluorocytosine).

The present invention encompasses pharmaceutically acceptable salts,acids or derivatives of the agents (and members of the classes ofagents) set forth above.

Dosing

The A_(2A)R/A_(2B)R inhibitors of the present invention may beadministered to a subject in an amount that is dependent upon, forexample, the goal of administration (e.g., the degree of resolutiondesired); the age, weight, sex, and health and physical condition of thesubject to which the formulation is being administered; the route ofadministration; and the nature of the disease, disorder, condition orsymptom thereof. The dosing regimen may also take into consideration theexistence, nature, and extent of any adverse effects associated with theagent(s) being administered. Effective dosage amounts and dosageregimens can readily be determined from, for example, safety anddose-escalation trials, in vivo studies (e.g., animal models), and othermethods known to the skilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (themaximum tolerated dose (MTD)) and not less than an amount required toproduce a measurable effect on the subject. Such amounts are determinedby, for example, the pharmacokinetic and pharmacodynamic parametersassociated with ADME, taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the A_(2A)R/A_(2B)R inhibitors of thepresent invention may be an amount that, when administered in one ormore doses to a subject, produces a desired result relative to a healthysubject. For example, for a subject experiencing a particular disorder,an effective dose may be one that improves a diagnostic parameter,measure, marker and the like of that disorder by at least about 5%, atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or more than90%, where 100% is defined as the diagnostic parameter, measure, markerand the like exhibited by a normal subject.

In certain embodiments, the A_(2A)R/A_(2B)R inhibitors contemplated bythe present invention may be administered (e.g., orally) at dosagelevels of about 0.01 mg/kg to about 50 mg/kg, or about 1 mg/kg to about25 mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic effect.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient.

In certain embodiments, the dosage of the desired A_(2A)R/A_(2B)Rinhibitor is contained in a “unit dosage form”. The phrase “unit dosageform” refers to physically discrete units, each unit containing apredetermined amount of the A_(2A)R/A_(2B)R inhibitor, either alone orin combination with one or more additional agents, sufficient to producethe desired effect. It will be appreciated that the parameters of a unitdosage form will depend on the particular agent and the effect to beachieved.

Kits

The present invention also contemplates kits comprising a compounddescribed herein, and pharmaceutical compositions thereof. The kits aregenerally in the form of a physical structure housing variouscomponents, as described below, and may be utilized, for example, inpracticing the methods described above.

A kit can include one or more of the compounds disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. Thecompounds described herein can be provided in a form that is ready foruse (e.g., a tablet or capsule) or in a form requiring, for example,reconstitution or dilution (e.g., a powder) prior to administration.When the compounds described herein are in a form that needs to bereconstituted or diluted by a user, the kit may also include diluents(e.g., sterile water), buffers, pharmaceutically acceptable excipients,and the like, packaged with or separately from the compounds describedherein. When combination therapy is contemplated, the kit may containthe several agents separately or they may already be combined in thekit. Each component of the kit may be enclosed within an individualcontainer, and all of the various containers may be within a singlepackage. A kit of the present invention may be designed for conditionsnecessary to properly maintain the components housed therein (e.g.,refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, tube or vial).

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the interne, are provided.

Experimental

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent that the experiments below were performed or thatthey are all of the experiments that may be performed. It is to beunderstood that exemplary descriptions written in the present tense werenot necessarily performed, but rather that the descriptions can beperformed to generate data and the like of a nature described therein.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.), but some experimental errors anddeviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: wt=wildtype; bp=base pair(s);kb=kilobase(s); nt=nucleotides(s); aa=amino acid(s); s or sec=second(s);min=minute(s); h or hr=hour(s); ng=nanogram; μg=microgram; mg=milligram;g=gram; kg=kilogram; dl or dL=deciliter; μl or μL=microliter; ml ormL=milliliter; 1 or L=liter; μM=micromolar; mM=millimolar; M=molar;kDa=kilodalton; i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC orSQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PBS=phosphate-buffered saline;IHC=immunohistochemistry; DMEM=Dulbeco's Modification of Eagle's Medium;EDTA=ethylenediaminetetraacetic acid.

Materials and Methods

The following general materials and methods were used, where indicated,or may be used in the Examples below:

Standard methods in molecular biology are described in the scientificliterature (see, e.g., Sambrook and Russell (2001) Molecular Cloning,3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;and Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloningin bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammaliancells and yeast (Vol. 2), glycoconjugates and protein expression (Vol.3), and bioinformatics (Vol. 4)).

The scientific literature describes methods for protein purification,including immunoprecipitation, chromatography, electrophoresis,centrifugation, and crystallization, as well as chemical analysis,chemical modification, post-translational modification, production offusion proteins, and glycosylation of proteins (see, e.g., Coligan, etal. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wileyand Sons, Inc., NY).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments,are available (see, e.g.,GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); and DeCypher™(TimeLogic Corp., Crystal Bay, Nev.).

The literature is replete with assays and other experimental techniquesthat can serve as a basis for evaluation of the compounds describedherein.

EXAMPLES

General Methods for Preparation of Compounds of the Claims

Those skilled in the art will recognize that there are a variety ofmethods available to prepare molecules represented in the claims. Ingeneral, useful methods for synthesizing compounds represented in theclaims consist of four parts, which may be done in any order: Connectionof the a and b fragments (or formation of the a-b-c moiety via b ringcyclization), connection of the b and c fragments (or formation of thea-b-c moiety via b ring cyclization), and modification of the functionalgroups present in all fragments. Retrosynthetic disconnection of thecompounds of the invention into fragments a-c useful for construction ofthe compounds is shown below:

Several methods for the preparation of claimed compounds are exemplary(eq. 1-5). Equation 1 demonstrates one method of synthesizing anappropriately functionalized fragment c. In the case of eq. 1, readilyavailable 2-aminobenzoic acids are converted to quinazolines viacondensation with urea followed by treatment with phosphoryl chloride.

Alternatively, a wide variety of methods are known in the art for theformation of quinazoline and quinoline rings (see for instance Joule etal., “Heterocyclic Chemistry”, Chapman & Hall, New York, or “Synthesisof Quinazolines” inhttp://www.organic-chemistry.org/synthesis/heterocycles/benzo-fused/quinazolines.shtm).

Equation two demonstrates one method of forming the bond betweenfragments b and c via a Suzuki reaction. In the case of eq. 2, Z may bechosen from an appropriate group such as Cl, Br, I, OTf, etc., and—B(OR)2 is a boronic acid or ester and the coupling is mediated by atransition metal catalyst, preferably palladium with an appropriateligand.

The coupling may be assisted by the use of an organic or inorganic base,and a wide variety of conditions are known in the art to facilitate theSuzuki coupling. The functionalization of the coupling partners may alsobe reversed as exemplified in eq. 3. Those skilled in the art willrecognize that there are other possible combinations which will alsoresult in the desired product. Formation of the bond between the b and cfragments may take place before or after formation of the connectionbetween the a and b fragments, and the groups may be further modifiedbefore or after connection of the c and b fragments.

Alternatively, the b fragment may be formed by cycloaddition between thea and c fragments via an azide-alkyne Huisgen 1,3-dipolar cycloaddition(Equation four). In the case of eq. 4, the appropriately functionalizeda and c fragments may be combined together in the cycloaddition reactionbetween an azide and an alkyne. The reaction may be facilitated via theuse of a copper catalyst or other catalyst.

In the case where fragment b is a triazole, the ring may also besynthesized via a palladium mediated addition of sodium azide to alkenylhalides (Barluenga et. al., Angew. Chem. Int. Ed., 2006, 45, 6893-6896),the Amberlyst-15 catalyzed addition of an azide to a nitroalkene (Zhanget. al., Synthesis, 2016, 48, 131-135), the I₂/TBPB mediated oxidativecycloaddition of N-tosylhydrozones with anilines (Cai et. al., Org.Lett., 2014, 16, 5108-5111), and a host of other methods (see “Synthesisof 1,2,3-triazoles” inwww.organic-chemistry.org/synthesis/heterocycles/1,2,3-triazoles.shtm).One skilled in the art will understand that there are a wide variety ofmethods available to effect this transformation.

Equation five demonstrates one method of forming the bond betweenfragments a and b via alkylation. In the case of eq. 5, Z is anappropriate electrophile such as Cl, Br, I, OTf, etc. and the couplingis mediated via an organic or inorganic base. For the most efficientpreparation of any particular compound of the invention, one skilled inthe art will recognize that the timing and the order of connection ofthe fragments and modification of the functionality present in any ofthe fragments may vary in the preparation of any given compound.

A variety of the methods described above have been used to preparecompounds of the invention, some of which are exemplified in theexamples.

Example 1 Synthesis of2-(6-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

Step 1: 250 mg (1.19 mmol) of commercially available4-chloro-8-methoxyquinazolin-2-amine in DMF and Et₃N (2 mL, 1:1) waspurged with nitrogen for 2 min. Trimethylslilylacetylene (234 mg, 2.38mmol), CuI (23 mg, 0.19 mmol) and Pd(PPh₃)₂Cl₂ (42 mg, 0.059) were addedsuccessively and the reaction mixture was heated at 90° C. for 2 h, Et₃Nwas removed on rotavapor and the residue was diluted with water, theseparated dark brown color solid was filtered and dried (320 mg, crude)

Step 2: The above crude TMS product was dissolved in 4 mL dry THF andcooled to 0° C. To this was added 1.2 mL (1.2 mmol) of TBAF (1.0 M inTHF). The reaction mixture was stirred at 0° C. for 15 min. SaturatedNH₄Cl (5 mL) was added to quench the reaction. The organics wereextracted from the aqueous layer with EtOAc (2×10 mL). The combinedorganic layer was washed with (1:1) NH₄Cl/NH₄OH (2×5 mL). The organiclayer was dried using Na₂SO₄, concentrated and the crude product (170mg) was used as such in the next step.

Step 3: To a solution of methylmagnesium bromide (3 M in Et₂O, 40 mL,120 mmol, 4.0 equiv) at 0° C. under N₂ was added a solution of methyl2-(hydroxymethyl)pyridine-2-carboxylate (5.0 g, 29.9 mmol) in THF (70mL, 0.4 M) over the course of 30 minutes. The resulting mixture wasallowed to warm to room temperature and stirred for 3 h. The reactionmixture was quenched with NH₄Cl aq (55 mL) and EtOAc (50 mL) was added.The organic phase was separated, and the aqueous phase was extractedwith EtOAc (3×40 mL). The combined organic extracts were washed withsaturated aqueous sodium bisulfite (7×20 mL), then dried (Na₂SO₄),filtered and concentrated in vacuo to give the title compound (3.45 g,69% yield; 96% purity as judged by LCMS) as a pale yellow liquid. LCMS:Method A, retention time=0.722 and 1.06 min, ESI MS [M+H]⁺ for C₉H₁₃NO₂,calcd 167.09, found 167.2

Step 4: To a solution of2-hydroxymethyl-6-(1-hydroxy-1-methylethyl)pyridine (5 g, 29.9 mmol, 1.0equiv) in PhMe (33 mL, 0.9 M) at 0° C. under N₂ was addeddiphenylphosphoryl azide (7.73 mL, 35.9 mmol, 1.2 equiv.), followed by1,8-diazabicyclo[5.4.0]undec-7-ene (5.37 mL, 35.9 mmol, 1.2 equiv.). Theresulting mixture was to warm to room temperature and stirred for 14 h.Upon completion, diluted with ethyl acetate and washed with water, theorganic layer was dried (Na₂SO₄), filtered and concentrated. The residuewas dissolved in 1N aq HCl (2 eq, 60 mmol) and extracted with MTBE inhexanes (3:7, 100 mL), the organic layer was washed with water (50 mL)and the combined aqueous layer was neutralized with 2N aqueous NaOH andextracted with ethyl acetate (3×75 mL), dried the organic layer(Na₂SO₄), filtered through a plug of cotton and concentrated thefiltrate to afford the pure compound as pale yellow color liquid (3.75g, 75%). LCMS: retention time=2.67 min, ESI MS [M+H]⁺ for C₉H₁₂N₄O,calcd 193.1, found 193.2

Step 5: A mixture of azide from step 4 (39 mg, 0.2 mmol), and alkynefrom step 2 (40 mg, 0.2 mmol), copper(II) sulfate (2.5 mg; 0.01 mmol),and sodium ascorbate (8 mg, 0.04 mmol) in 2:1 t-BuOH/H₂O (2 mL) washeated at 60° C. for 2 h. The solvent was removed in vacuo, the residuedry loaded onto silica gel, and purified by silica gel chromatography(0-20% methanol in dichloromethane) to afford the desired product as ayellow solid (46 mg, 58%). δ ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (s, 1H),8.58-8.49 (m, 1H), 7.84-7.75 (m, 1H), 7.59 (d, J=7.9 Hz, 1H), 7.19-7.09(m, 3H), 6.83 (bs, 2H), 5.84 (s, 2H), 5.21 (s, 1H), 3.87 (s, 3H), 1.36(s, 6H). ESI MS [M+H]⁺ for C₂₀H₂₁N₇O₂, calcd 392.2, found 392.4.

Example 2 Synthesis of4-{1-[(6-tert-butylpyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}-8-methoxyquinazolin-2-amine

The title compound was synthesized in a similar fashion to example 1. ¹HNMR (400 MHz, Chloroform-d) δ 8.81 (dd, J=8.6, 1.3 Hz, 1H), 8.58 (d,J=1.2 Hz, 1H), 7.62 (td, J=7.8, 1.3 Hz, 1H), 7.48-7.14 (m, 2H),7.14-7.00 (m, 2H), 5.73 (s, 2H), 5.22 (s, 2H), 4.04 (s, 3H), 1.36 (s,9H). ESI MS [M+H]⁺ for C₂₁H₂₂N₇O, calcd 390.2, found 390.3.

Example 3 Synthesis of8-methoxy-4-(1-{[6-(methoxymethyl)pyridin-2-yl]methyl}-1H-1,2,3-triazol-4-yl)quinazolin-2-amine

The title compound was synthesized in a similar fashion to example 1. ¹HNMR (400 MHz, Chloroform-d) δ 8.85 (dt, J=8.5, 1.3 Hz, 1H), 8.49 (d,J=1.4 Hz, 1H), 7.85-7.59 (m, 1H), 7.42 (d, J=7.8 Hz, 1H), 7.33-7.16 (m,1H), 7.14 (dd, J=18.5, 7.7 Hz, 2H), 5.76 (s, 2H), 5.22 (s, 2H), 4.67 (s,2H), 4.04 (s, 3H), 3.50 (s, 3H). ESI MS [M+H]⁺ for C₁₉H₁₉N₇O₂, calcd378.2, found 378.2.

Example 4 Synthesis of4-{1-[(6-cyclopropylpyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}-8-methoxyquinazolin-2-amine

The title compound was synthesized in a similar fashion to example 1. ¹HNMR (400 MHz, DMSO-d₆) δ 8.74 (s, 1H), 8.54 (s, 1H), 7.66 (t, J=7.8 Hz,1H), 7.19 (dd, J=23.9, 6.3 Hz, 3H), 7.04 (d, J=7.6 Hz, 1H), 6.84 (s,2H), 5.77 (s, 2H), 3.87 (s, 3H), 2.05 (d, J=8.0 Hz, 1H), 0.89 (d, J=7.9Hz, 2H), 0.80 (s, 2H). ESI MS [M+H]⁺ for C₂₀H₁₉N₇O, calcd 373.2, found374.2.

Example 5 Synthesis of4-{1-[(6-cyclopropylpyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}-8-methoxyouinazolin-2-amine

Step 1: A solution of n-butyllithium (144 mL, 360 mmol, 2.5 M inhexanes) in ether (120 mL) was cooled to −78° C. and2-bromo-6-methylpyridine (41.0 mL, 360 mmol) was added dropwise. Thereaction mixture was warmed to 0° C. and stirred at this temperature for15 minutes. In a separate flask dibutyl sulfide (54.5 mL, 312 mmol) andcopper(I) iodide (34.3 g, 180 mmol) were combined and the mixturestirred for 5 minutes until homogeneous. Ether (240 mL) was added, thesolution cooled to 0° C., and the pyridine solution from above was addeddropwise. The mixture was stirred for an additional 20 minutes at 0° C.at which point a solution of 1(16.7 g, 120 mmol) in ether (120 mL) wasadded. The reaction mixture was allowed to warm to room temperature over14 hours. The mixture was quenched with saturated ammonium chloridesolution and extracted ethyl acetate (2×200 mL), washed with brine, anddried over sodium sulfate. The crude product was purified by silica gelchromatography (0 to 20% EtOAc in hexanes) to afford the desired product2 as a brown oil (16.44 g; 59%).

Step 2: A mixture of 2 (16.44 g, 70.8 mmol), sodium chloride (1.24 g,21.2 mmol), water (1.42 mL), and DMSO (71 mL) were heated at 160° C. for3 hours. The reaction mixture was cooled, MTBE (500 mL) was added, theorganic phase washed with water (4×400 mL), and dried over sodiumsulfate. The crude material was dissolved in 3.0 M methanolic HCl (236mL) and heated at 50° C. for 60 h. The reaction mixture was slowlyquenched with sodium bicarbonate(,), filtered, and concentrated. Thecrude product was purified by silica gel chromatography (7.5% EtOAc inhexanes) to afford the desired product 3 as a colorless oil (8.73 g;59%).

Step 3: To a solution of 3 (8.73 g, 42.1 mmol) in DCM (168 mL) at 0° C.was added m-CPBA (19.9 g, 84.2 mmol, 75% in water) slowly as a solidover 5 minutes. The reaction mixture was stirred at 0° C. for 1 hour andat room temperature for 14 hours. The organic layer was washed with 0.1M NaOH solution, dried over sodium sulfate, and concentrated. The crudematerial was re-dissolved in DCM (84 mL), cooled to 0° C., and TFAA (59mL) was added dropwise. The reaction mixture was stirred at roomtemperature for 3 hours. The reaction mixture was slowly quenched with asaturated Na₂CO₃ solution and extracted with ethyl acetate (3×200 mL).The crude material was purified by silica gel chromatography (0 to 75%EtOAc in hexanes) to afford the desired product 4 as a red oil (5.55 g;59%).

Step 4: To a mixture of 4 (5.55 g, 24.9 mmol), DPPA (6.42 g, 29.8 mmol),and toluene (25 mL) was added DBU (4.46 mL, 29.8 mmol). The reactionmixture was stirred at room temperature for 14 hours. The mixture waspurified by silica gel chromatography (0 to 20% EtOAc in hexanes) toafford the desired product 5 as a colorless oil (5.53 g; 89%).

Step 5: Copper sulfate mediated cycloaddition was carried out in asimilar fashion to example 1 and saponification of the ester with LiOHafforded the title compound. ¹H NMR (400 MHz, DMSO-d₆) δ 11.80 (s, 1H),8.77 (s, 1H), 8.53 (t, J=4.9 Hz, 1H), 7.84-7.67 (m, 1H), 7.37 (d, J=7.9Hz, 1H), 7.25-7.11 (m, 1H), 7.08 (d, J=7.6 Hz, 1H), 6.82 (s, 2H), 5.83(s, 1H), 3.87 (s, 3H), 2.64 (s, 2H), 1.31 (s, 6H). ESI MS [M+H]⁺ forC₂₂H₂₃N₇O₃, calcd 434.2, found 434.3

Example 6 Synthesis of8-methoxy-4-{1-1(6-{[(3S)-oxolan-3-yloxy]methyl}pyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}quinazolin-2-amine

Step 1: A round-bottom flask was charged with 1.8 g (7.8 mmol) ofdichloro-quinazoline, 273.8 mg (0.39 mmol) of PdCl₂(PPh₃)₂ and 148.2 mg(0.78 mmol) of CuI. The content was vacuum degassed and backfilled withN₂ three times. 40 mL of degassed THF was added to the flask followed byaddition of 3.35 mL (24 mmol) of degassed Et₃N and 1.75 mL (7.8 mmol) ofdegassed TIPS-acetylene. The reaction mixture was stirred at roomtemperature for 6 hours under N₂. Then the reaction mixture diluted with50 mL EtOAc, transferred to a separatory funnel and subsequently washedwith (1:1) NH₄Cl/NH₄OH (2×50 mL) and brine (1×50 mL). The organic layerwas dried over Na₂SO₄, concentrated and purified by silica gelchromatography eluting with 10% EtOAc/Hexane to give 2.79 g (96%) of theTIPS product.

Step 2: A 40-mL screwcap vial was charged with 440 mg (1.2 mmol) ofabove 2-chloroquinazoline derivative and 2 mL of 4-methoxy-benzylamine.The vial was sealed and heated at 100° C. for 2 hours. After cooling toroom temperature, 30 mL of EtOAc was added and the reaction wastransferred to a separatory funnel. The diluted reaction mixture wassuccessively washed with water (2×25 mL) and 10% aqueous citric acid(2×25 mL). The organic layer was dried over Na₂SO₄, concentrated andpurified by silica gel chromatography eluting with 20% EtOAc/Hexane togive 450 mg (79%) of PMB derivative.

Step 3: A 40-mL screwcap vial was charged with 450 mg (0.95 mmol) ofTIPS-acetylene derivative 3. To this vial was added 3.5 mL of THF and0.2 mL of H₂O and stirred at room temperature. After 3 has completelydissolved, it was cooled to 0° C. and 125 μL (0.19 mmol) of 40% nBu₄NOHwas added. The ice-bath was removed and the reaction was stirred at roomtemperature for 15 min. The reaction was quenched with 10 mL saturatedNH₄Cl. The aqueous layer was extracted with EtOAc (2×30 mL), dried overNa₂SO₄ and concentrated. Trituration with 40% CH₂Cl₂/Hexane gave purealkyne as yellow solid (258 mg, 85%)

Step 4: To a 0° C. stirred solution of the 3(S)-hydroxytetrahydrofuran(440 mg, 5 mmol) in dry THF (20 mL) was added NaH (60%, 400 mg, 10 mmol)in 5 portions. It was stirred at this temperature for 30 min. A graycolor suspension was obtained, to this reaction mixture was added2,6-bis(chloromethyl)pyridine hydrochloride (1.06 g, 5 mmol) in oneportion at 0° C. The reaction mixture was stirred at room temperaturefor overnight. It was cooled to 0° C., quenched with saturated aqueousNH₄Cl solution, diluted with MTBE (10 mL), the layers were separated,aqueous layer was extracted with MTBE and the organics were combined,dried (Na₂SO₄), filtered and concentrated on rotavapor. The oily residuewas dissolved in dichloromethane purified by flash column (ISCO, 40 gcolumn,5-60% ethyl acetate in hexanes) to get the pure compound ascolorless liquid (480 mg, 42%).

Step 5: The above product (480 mg, 2.1 mmol) was dissolved in dry DMSO(2 mL), NaN₃ (164 mg, 2.53 mmol) was added and stirred at r.t for 2hours. LCMS indicated completion of the reaction, it was diluted withwater (15 mL), extracted with MTBE (3×15 mL), dried (Na₂SO₄), filtered,and concentrated on rotavapor. The oily residue was dried under highvacuum to afford the product (455 mg, 92%).

Step 6: Alkyne (example 6a, 80 mg, 0.25 mmol, 1 equiv.) and azide(example 6b, 59 mg, 0.25 mmol, 1 equiv.) were dissolved in CH₂Cl₂ (0.5mL) and a 2:1 mixture of tBuOH/H₂O (1 mL). CuSO₄·5H₂O (3.1 mg, 0.013mmol, 5 mol %) and sodium ascorbate (9.9 mg, 0.05 mmol, 20 mol %) wereadded, and the mixture was stirred until the starting material wasconsumed as determined by LCMS analysis. The reaction mixture wasdirectly purified by flash chromatography on SiO₂ (0-20% MeOH/CH₂Cl₂) toafford PMB derivative (138 mg, quantitative) as a yellow oil. It wasdissolved in TFA (1 mL), and the reaction mixture was placed in aheating block preheated to 70° C. overnight. The reaction was cooled toroom temperature and concentrated under a stream of nitrogen. NaHCO₃ wasadded to neutralize residual TFA, CH₂Cl₂ was added, and the layers wereseparated. The organic extracts were concentrated, and the crude residuewas purified by flash chromatography on SiO₂ (0-20% MeOH/CH₂Cl₂) toafford (95 mg, 88%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80(d, J=1.7 Hz, 1H), 8.59 (ddd, J=5.9, 3.9, 1.8 Hz, 1H), 7.87 (td, J=7.8,1.9 Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.23-7.10(m, 2H), 6.86 (s, 2H), 5.87 (d, J=1.9 Hz, 2H), 4.52 (d, J=1.8 Hz, 2H),4.24 (qd, J=3.7, 2.9, 1.8 Hz, 1H), 3.89 (d, J=1.7 Hz, 3H), 3.80-3.60 (m,4H), 1.94 (tdt, J=7.9, 5.2, 2.2 Hz, 2H). ESI MS [M+H]⁺ for C₂₂H₂₃N₇O₃,calcd 434.2, found 434.3

Example 7 Synthesis of8-methoxy-4-{1-[(6-{[(3R)-oxolan-3-yloxy]methyl}pyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}quinazolin-2-amine

The title compound was synthesized in a similar fashion to example 6 toafford 94 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d,J=1.7 Hz, 1H), 8.59 (ddd, J=5.9, 3.9, 1.8 Hz, 1H), 7.87 (td, J=7.8, 1.9Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.23-7.10 (m,2H), 6.86 (s, 2H), 5.87 (d, J=1.9 Hz, 2H), 4.52 (d, J=1.8 Hz, 2H), 4.24(qd, J=3.7, 2.9, 1.8 Hz, 1H), 3.89 (d, J=1.7 Hz, 3H), 3.80-3.60 (m, 4H),1.94 (tdt, J=7.9, 5.2, 2.2 Hz, 2H). ESI MS [M+H]⁺ for C₂₂H₂₃N₇O₃, calcd434.2, found 434.3.

Example 8 Synthesis of8-methoxy-4-{1-1(6-{[(3R)-oxolan-3-yloxy]methyl}pyridin-2-yl)methyl]-1H-1,2,3-triazol-4-yl}quinazolin-2-amine

The title compound was synthesized in a similar fashion to example 6 toafford 46 mg of a yellow wax. ¹H NMR (400 MHz, CDCl₃) δ 8.91-8.81 (m,1H), 8.55-8.44 (m, 1H), 7.72 (t, J=7.7 Hz, 1H), 7.48 (d, J=7.7 Hz, 1H),7.35-7.21 (m, 1H), 7.14 (dd, J=19.8, 7.7 Hz, 2H), 5.75 (d, J=1.9 Hz,2H), 5.24 (s, 2H), 4.71 (d, J=1.9 Hz, 2H), 4.12-3.98 (m, 3H), 3.74 (dt,J=5.6, 2.3 Hz, 2H), 3.62 (dt, J=5.9, 3.1 Hz, 2H), 3.48-3.33 (m, 3H). ESIMS [M+H]⁺ for C₂₁H₂₃N₇O₃, calcd 422.2, found 422.3.

Example 9 Synthesis of1-[(6-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)methyl]pyrrolidin-2-one

The title compound was synthesized in a similar fashion to example 6. ¹HNMR (400 MHz, DMSO-d₆) δ 8.78 (s, 1H), 8.60 (dd, J=6.2, 3.5 Hz, 1H),7.82 (t, J=7.8 Hz, 1H), 7.28 (d, J=7.6 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H),7.18-7.11 (m, 2H), 6.83 (s, 2H), 5.87 (s, 2H), 4.41 (s, 2H), 3.87 (s,3H), 3.21 (t, J=7.0 Hz, 2H), 2.17 (t, J=8.0 Hz, 2H), 1.78 (p, J=7.6 Hz,2H). ESI MS [M+H]⁺ for C₂₂H₂₂N₈O₂, calcd 331.2, found 331.2.

Example 10 Synthesis of2-(6-{[4-(2-amino-6-fluoroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 6. ¹HNMR (400 MHz, DMSO-d₆) δ 8.90-8.75 (m, 2H), 7.80 (t, J=7.8 Hz, 1H),7.70-7.62 (m, 1H), 7.60 (d, J=8.0 Hz, 1H), 7.53 (ddd, J=9.3, 5.4, 1.4Hz, 1H), 7.15 (d, J=7.7 Hz, 1H), 6.83 (s, 2H), 5.97-5.75 (m, 2H), 5.21(s, 1H), 1.35 (s, 6H). ESI MS [M+H]⁺ for C₁₉H₁₈N₇O, calcd 380.2, found380.1.

Example 11 Synthesis of2-(6-{[4-(2-amino-5-fluoroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 6. ¹HNMR (400 MHz, DMSO-d₆) δ 8.61 (s, 1H), 7.84-7.77 (m, 1H), 7.71-7.63 (m,1H), 7.60 (d, J=8.2 Hz, 1H), 7.32 (d, J=8.5 Hz, 1H), 7.15 (s, 2H), 7.05(s, 1H), 6.91 (dd, J=11.3, 7.9 Hz, 1H), 5.79 (s, 2H), 5.22 (s, 1H), 1.39(s, 6H). ESI MS [M+H]⁺ for C₁₉H₁₈N₇O, calcd 380.2, found 380.1.

Example 12 Synthesis of2-(6-{[4-(2-amino-7-fluoro-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 6 toafford 100 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.99 (s,1H), 8.91 (s, 1H), 7.82 (td, J=7.8, 1.0 Hz, 1H), 7.62 (d, J=7.8 Hz, 1H),7.34 (s, 1H), 7.18 (d, J=7.4 Hz, 1H), 5.90 (s, 2H), 4.04 (s, 3H), 1.36(d, J=1.3 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₂₀FN₇O₂, calcd 410.2, found410.3.

Example 13 Synthesis of2-(6-{[4-(2-amino-8-methylquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

Step 1: A solution of 3-methylanthranilic acid (3.78 g, 25 mmol, 1equiv.) in THF (25 mL) was cooled in an ice/water bath.Carbonyldiimidazole (4.87 g, 30 mmol, 1.2 equiv.) was added in oneportion. The reaction mixture stirred for two hours, and the resultingprecipitate was filtered, washed with MTBE, and dried under high vacuumto afford compound (3.13 g, 71%) as a tan solid.

Step 2: A suspension of the above isatoic anhydride derivative (3.13 g,17.7 mmol, 1 equiv.), S-methylisothiourea hydroiodide (3.85 g, 17.7mmol, 1 equiv.), sodium carbonate (2.06 g, 19.5 mmol, 1.1 equiv.) inMeCN (53 mL) and H₂O (13 mL) was placed in a heating block preheated to110° C. for 75 minutes. The reaction mixture was cooled to roomtemperature and left to stir for one hour, at which point theprecipitate was filtered and washed with a 4:1 mixture of MeCN/H₂O anddried under high vacuum to afford 2-aminoquinazolinone derivative (2.84g, 92%) as a greyish solid.

Step 3: A suspension of the above 2-aminoquinazolinone derivative (2.84g, 16.2 mmol, 1 equiv.) in POCl₃ (37 mL, 405 mmol, 25 equiv.) was placedin a heating block preheated to 90° C. for 4.5 hours. POCl₃ wasdistilled out, CH₂Cl₂ was added, and the mixture was cooled in anice/water bath. 4M KOH was added until the pH was basic, the layers wereseparated, and the organic layer was concentrated to yield impure4-chloro-2-aminoquinazoline derivative which was used without furtherpurification.

Step 4: The above crude 4-chloro-2-aminoquinazoline derivative (ca. 9.9mmol), trimethylsilylacetylene (4.1 mL, 29.5 mmol), Et₃N (4.1 mL, 29.5mmol), and THF (50 mL) were combined in a flask under N₂. Pd(PPh₃)₂Cl₂(351 mg, 0.5 mmol) and CuI (190 mg, 1 mmol) were added and the flask wasplaced in a heating block preheated to 80° C. for 90 minutes. Thereaction mixture was cooled to room temperature and filtered through aplug of Celite. The filtrate was concentrated onto ca. 10 g Celite andpurified by flash chromatography on SiO₂ (0-25% EtOAc/Hexanes) to affordTMS alkyne derivative (542 mg, 13%) as a tan solid.

Step 5: To a suspension of above TMS alkyne derivative (542 mg, 2.12mmol) in MeOH (10 mL) was added NH₃ solution (7 M in MeOH, 0.54 mL) atroom temperature. After one hour, the reaction mixture was concentratedto afford terminal alkyne derivative (388 mg, quantitative) as abrownish solid.

Step 6: The terminal alkyne derivative (46 mg, 0.25 mmol, 1 equiv.) andazide (from example 1, 48 mg, 0.25 mmol, 1 equiv.) were dissolved inCH₂Cl₂ (0.5 mL) and a 2:1 mixture of tBuOH/H₂O (1 mL). CuSO₄·5H₂O (3.1mg, 0.013 mmol, 5 mol %) and sodium ascorbate (9.9 mg, 0.05 mmol, 20 mol%) were added, and the mixture was stirred until the starting materialwas consumed as determined by LCMS analysis. The reaction mixture wasdirectly purified by flash chromatography on SiO₂ (0-20% MeOH/CH₂Cl₂) toafford target compound (84 mg, 89%) as a tan solid. ¹H NMR (400 MHz,CDCl₃) δ 9.07 (d, J=8.5 Hz, 1H), 8.47 (d, J=0.8 Hz, 1H), 7.82-7.67 (m,1H), 7.58 (dd, J=7.0, 1.8 Hz, 1H), 7.38 (d, J=7.9 Hz, 1H), 7.26-7.24 (m,1H), 7.17 (d, J=7.7 Hz, 1H), 5.79 (s, 2H), 5.09 (s, 2H), 2.69-2.50 (m,3H), 1.55 (d, J=0.8 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₂₁N₇O, calcd 375.2,found 375.3.

Example 14 Synthesis of2-[6-({4-[2-amino-8-(trifluoromethyl)quinazolin-4-yl1-1H-1,2,3-triazol-1-yl}methyl)pyridin-2-yl]propan-2-ol

The title compound was synthesized in a similar fashion to example 13 toafford 60 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.32 (dd,J=8.5, 1.7 Hz, 1H), 8.88 (d, J=1.5 Hz, 1H), 8.16-8.06 (m, 1H), 7.82 (td,J=7.8, 1.4 Hz, 1H), 7.62 (dt, J=7.9, 1.2 Hz, 1H), 7.37 (t, J=8.0 Hz,1H), 7.25-7.12 (m, 3H), 5.89 (d, J=1.4 Hz, 2H), 5.23 (d, J=1.5 Hz, 1H),1.37 (d, J=1.4 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₁₈F₃N₇O, calcd 430.2,found 430.3.

Example 15 Synthesis of2-(6-{[4-(2-aminoquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 13 toafford 43 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 9.02 (t,J=8.0 Hz, 1H), 8.91-8.76 (m, 1H), 7.81 (t, J=7.7 Hz, 1H), 7.76-7.67 (m,1H), 7.62 (t, J=7.6 Hz, 1H), 7.49 (t, J=7.9 Hz, 1H), 7.34-7.23 (m, 1H),7.16 (t, J=7.5 Hz, 1H), 6.81 (s, 2H), 5.88 (d, J=7.0 Hz, 2H), 5.28-5.21(m, 1H), 1.46-1.28 (m, 6H). ESI MS [M+H]⁺ for C₁₉H₁₉N₇O, calcd 362.2,found 362.3.

Example 16 Synthesis of2-[6-({4-[2-amino-8-(trifluoromethoxy)quinazolin-4-yl]-1H-1,2,3-triazol-1-yl}methyl)pyridin-2-yl]propan-2-ol

The title compound was synthesized in a similar fashion to example 13.¹H NMR (400 MHz, DMSO-d₆) δ 9.07 (d, J=8.5 Hz, 1H), 8.87 (s, 1H), 7.82(t, J=7.8 Hz, 1H), 7.77 (d, J=7.7 Hz, 1H), 7.62 (d, J=7.9 Hz, 1H),7.35-7.26 (m, 1H), 7.20 (br s, 2H), 7.17 (d, J=8.7 Hz, 1H), 5.89 (s,2H), 5.23 (s, 1H), 1.37 (s, 6H). ESI MS [M+H]⁺ for C₂₀H₁₉F₃N₇O₂, calcd446.2, found 446.2.

Example 17 Synthesis of2-(6-{[4-(2-amino-8-fluoroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 13.¹H NMR (400 MHz, DMSO-d₆) δ 8.91-8.84 (m, 2H), 7.81 (t, J=7.8 Hz, 1H),7.62 (d, J=8.0 Hz, 1H), 7.56 (ddd, J=11.2, 7.7, 1.3 Hz, 1H), 7.26-7.19(m, 1H), 7.16 (d, J=7.6 Hz, 1H), 7.12 (br s, 2H), 5.88 (s, 2H), 5.23 (s,1H), 1.37 (s, 6H). ESI MS [M+H]⁺ for C₁₉H₁₉FN₇O₂, calcd 380.2, found380.2.

Example 18 Synthesis of2-(6-{[4-(2-amino-7-fluoroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 13.¹H NMR (400 MHz, DMSO-d₆) δ 9.21-9.12 (m, 1H), 8.84 (s, 1H), 7.81 (td,J=7.8, 1.1 Hz, 1H), 7.62 (d, J=7.9 Hz, 1H), 7.22-7.12 (m, 3H), 6.98 (s,2H), 5.88 (s, 2H), 5.23 (s, 1H), 1.37 (s, 6H). ESI MS [M+H]⁺ forC₁₉H₁₉FN₇O₂, calcd 380.2, found 380.1

Example 19 Synthesis of2-(6-{[4-(2-amino-8-chloroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 13.¹H NMR (400 MHz, DMSO-d₆) δ 9.01 (d, J=8.4 Hz, 1H), 8.84 (s, 1H), 7.88(d, J=7.5 Hz, 1H), 7.80 (t, J=7.8 Hz, 1H), 7.60 (d, J=7.9 Hz, 1H),7.27-7.06 (m, 4H), 5.86 (s, 2H), 5.21 (s, 1H), 1.35 (s, 6H). ESI MS[M+H]⁺ for C₁₉H₁₈ClN₇O, calcd 396.1, found 396.2.

Example 20 Synthesis of2-(6-{[4-(2-amino-5-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 13 toafford 15 mg of a tan solid. ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J=1.5Hz, 1H), 7.74 (td, J=7.7, 1.6 Hz, 1H), 7.66-7.49 (m, 1H), 7.42-7.32 (m,1H), 7.19 (ddt, J=11.8, 7.7, 1.1 Hz, 2H), 6.56 (dd, J=8.0, 1.0 Hz, 1H),5.77 (d, J=1.8 Hz, 2H), 5.42 (s, 2H), 3.59 (d, J=1.7 Hz, 3H), 1.53 (d,J=1.7 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₂₁N₇O₂, calcd 392.2, found 392.3.

Example 21 Synthesis of1-(6-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)cyclobutan-1-ol

Step 1: A round-bottom flask was charged with 2.0 g (6.7 mmol) ofcommercially available 2-bromo-pyridine derivative. To this flask wasadded 13.0 mL of dry THF and cooled to −78° C. under N₂. nBuLi 2.7 mL(2.5 M in THF) was added dropwise to the reaction at −78° C. and stirredfor 30 min. Cyclobutanone (0.58 mL, 7.9 mmol) was then added inone-portion and the reaction warmed to room temperature over 2 h (LCMSshows formation of the desired addition product). The reaction mixturewas cooled back to 0° C. and 6.7 mL of TBAF (1 M in THF) was added.After stirring the reaction for 15 min at 0° C., 50.0 mL saturatedaqueous NH₄Cl was added to quench the reaction. The aqueous layer wasextracted with EtOAc (2×50 mL), dried over Na₂SO₄ and concentrated. Thecrude material was purified by silica gel chromatography to obtain diol2 (570 mg, 48% in 2 steps).

Step 2: To a solution of the product from step 1 (570 mg, 3.2 mmol) inCH₂Cl₂ (4.0 mL) was added diphenyl-phosphorylazide (0.8 mL, 3.8 mmol)and DBU (0.6 mL, 3.8 mmol) at room temperature. The reaction mixture wasstirred at room temperature for 10 h under N₂. After removing CH₂Cl_(2,)the residue was re-dissolved in EtOAc and subsequently washed with H₂O(2×25 mL). The organic layer was dried over Na₂SO₄ and concentrated. Thecrude material was purified by silica gel chromatography to obtain azidederivative (450 mg, 69%).

Step 3: The title compound was synthesized in a similar fashion toexample 6 to afford 78 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ8.97 (s, 1H), 8.80 (d, J=8.4 Hz, 1H), 7.82 (td, J=7.9, 1.5 Hz, 1H),7.57-7.38 (m, 3H), 7.26 (dd, J=7.6, 1.4 Hz, 1H), 5.96 (s, 2H), 4.01 (s,3H), 2.41-2.31 (m, 2H), 2.23-2.07 (m, 2H), 1.81-1.56 (m, 2H). ESI MS[M+H]⁺ for C₂₁H₂₁N₇O₂, calcd 404.2, found 404.3.

Example 22 Synthesis of1-(6-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)cyclopentan-1-ol

The title compound was synthesized in a similar fashion to example 21 toafford 67 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d,J=1.2 Hz, 1H), 8.61-8.51 (m, 1H), 7.90-7.72 (m, 1H), 7.63 (dd, J=7.9,1.2 Hz, 1H), 7.24-7.08 (m, 3H), 6.85 (s, 2H), 5.86 (s, 2H), 5.07 (s,1H), 3.97-3.80 (m, 3H), 2.67 (s, 1H), 1.95 (dd, J=19.3, 11.7 Hz, 2H),1.86-1.61 (m, 5H). ESI MS [M+H]⁺ for C₂₂H₂₃N₇O₂, calcd 418.2, found418.1.

Example 23 Synthesis of2-amino-4-(1-{[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl}-1H-1,2,3-triazol-4-yl)quinazoline-8-carbonitrile

Step 1: To a suspension of 3-cyano-2-fluoro-benzoic acid (8.4 g, 50.9mmol) in 50.0 mL dry CH₂Cl₂, was added 13.1 mL (153.0 mmol) oxalylchloride and 2-drops of DMF. The reaction mixture was then stirred underN₂ for 10 h. Excess oxalyl chloride and solvent was distilled off andthe residue was re-dissolved in 50.0 mL dry THF. This THF solution ofthe acid-chloride was then dropwise added to a solution ofguanidine-hydrochloride (24.4 g, 255.0 mmol) in 150.0 mL H₂O containingNaOH (13.3 g, 332.5 mmol) at 0° C. After complete addition, ice bath wasremoved and the reaction was stirred at room temperature for 2 h. Theaqueous layer was extracted with EtOAc (2×100 mL), dried over Na₂SO₄ andconcentrated. The crude product thus obtained was used in next stepwithout further purification.

Step 2: To a solution of the crude product from step 1 in MeCN (250 mL)was added K₂CO₃ (21 g, 151.9 mmol at room temperature. The reactionmixture was then heated at reflux for 10 h. After cooling the reactionmixture to room temperature, 150.0 mL H₂O was added. The aqueous layerwas extracted with EtOAc (2×100 mL), dried over Na₂SO₄ and concentrated.The crude product thus obtained was used in next step without furtherpurification. To the crude 2-aminoquinazolinone derivative in around-bottom flask, was added 62.0 mL (663.1 mmol) POCl₃. The resultingsuspension was heated at 100° C. for 1 h. Excess POCl₃ was distilled offand the residual POCl₃ was quenched carefully with ice-water. Theaqueous layer was extracted with EtOAc (2×100 mL), dried over Na₂SO₄ andconcentrated. The crude 2-amino-4-chloroquinazoline derivative thusobtained was used in the synthesis of target compound (steps 3 and 4) ina similar fashion to example 1. ¹H NMR (400 MHz, DMSO-d₆) δ 9.34 (d,J=8.4 Hz, 1H), 8.87 (s, 1H), 8.24 (d, J=7.3 Hz, 1H), 7.80 (t, J=7.8 Hz,1H), 7.60 (d, J=8.0 Hz, 1H), 7.50-7.29 (m, 3H), 7.15 (d, J=7.6 Hz, 1H),5.87 (s, 2H), 5.21 (s, 1H), 1.35 (s, 6H). ESI MS [M+H]⁺ for C₂₀H₁₈N₈O,calcd 387.2, found 387.2.

Example 24 Synthesis of2-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(2-hydroxypropan-2-yl)pyridin-1-ium-1-olate

Step 1: m-CPBA ( 75%, 507 mg, 2.2 mmol, 1.1 equiv.) was added in oneportion to a solution of azide (example 6b, 384 mg, 1 mmol, 1 equiv.) inCH₂Cl₂ (10 mL). The reaction mixture was stirred for 4.5 hours andconcentrated. The crude residue was purified by flash chromatography onSiO₂ (0-100% EtOAc/Hexanes) to afford N-oxide derivative (366 mg, 88%)as a yellow oil.

Step 2: The title compound was synthesized in a similar fashion toexample 6 to afford 73 mg of a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ8.94-8.85 (m, 1H), 8.66-8.50 (m, 1H), 7.72 (dd, J=8.1, 2.0 Hz, 1H),7.54-7.45 (m, 1H), 7.23-7.12 (m, 3H), 6.89 (s, 2H), 6.64 (d, J=1.4 Hz,1H), 5.97 (s, 2H), 3.89 (d, J=1.4 Hz, 3H), 1.60 (d, J=1.4 Hz, 6H). ESIMS [M+H]⁺ for C₂₀H₂₂N₇O₃, calcd 408.2, found 408.3.

Example 25 Synthesis of4-{1-[(6-tert-butylpyridin-2-yl)methyl]-1H-pyrazol-4-yl}-8-methoxyquinazolin-2-amine

Step 1: A round-bottom flask was charged with 1.4 g (7.7 mmol) ofcommercially available chloride derivative and 1.63 g (8.4 mmol) ofcommercially available boronate. To this mixture was added 40 mL of dryMeCN and 2.9 g (8.4 mmol) Cs₂CO₃. The reaction mixture was then heatedat 65° C. for 3 hours. After cooling the reaction to room temperature,CH₂Cl₂ was added and the solid was filtered off. The filtrate thusobtained was concentrated and used in next step without furtherpurification.

Step 2: A 40-mL screwcap vial was charged with 222 mg (0.65 mmol) of theabove boronate derivative and 100 mg (0.48 mmol) of commerciallyavailable 2-amino-4-chloro-8-methoxyquinazoline. To this was added 2.0mL of MeCN and 0.5 mL 2.0 M aqueous K₂CO₃. After flushing the contentwith N₂, 55 mg (0.05 mmol) of Pd(PPh₃)₄ was added. The vial was sealedand heated at 80° C. for 8 hours. After cooling to room temperature, 30mL of EtOAc was added and the reaction was transferred to a separatoryfunnel. The diluted reaction mixture was successively washed with water(2×25 mL) and brine (2×25 mL). The organic layer was dried over Na₂SO₄,concentrated and purified by silica gel chromatography to give 130 mg(70%) of the title compound. ¹H NMR (400 MHz, Chloroform-d) δ 8.26 (s,1H), 8.14 (s, 1H), 7.72 (d, J=8.4 Hz, 1H), 7.58 (t, J=7.8 Hz, 1H),7.29-7.23 (m, 1H), 7.20-7.15 (m, 1H), 7.07 (d, J=7.9 Hz, 1H), 6.96 (d,J=7.6 Hz, 1H), 5.50 (s, 2H), 5.27 (s, 2H), 4.03 (s, 3H), 1.36 (s, 9H).ESI MS [M+H]⁺ for C₂₂H₂₄N₆O, calcd 389.2, found 389.3.

Example 26 Synthesis of2-(6-{[4-(2-amino-8-methoxyquinazolin-4-yl)-1H-pyrazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

Step 1: To a solution of 2-(6-(hydroxymethyl)pyridin-2-yl)propan-2-ol(2.13 g, 12.7 mmol, 1.0 equiv) in CH₂Cl₂ (127 mL, 0.1 M) under N₂ wasadded CBr4 (4.7 g, 14.0 mmol, 1.1 equiv) followed by triphenylphosphine(3.7 g, 14.0 mmol, 1.1 equiv). The resulting mixture was stirred at roomtemperature for 4 h. Following this time, the reaction mixture wastransferred to a separatory funnel and washed with saturated aqueousNaHCO₃ (150 mL). The organic phase was collected, dried over MgSO₄, andconcentrated in vacuo. The resulting oil was purified by columnchromatography (CH₂Cl₂→9:1 CH₂Cl₂:MeOH) to give2-(6-(bromomethyl)pyridin-2-yl)propan-2-ol (2.0 g, 69% yield) as ayellow oil.

Step 2: 2-(6-(bromomethyl)pyridin-2-yl)propan-2-ol (1.0 g, 4.3 mmol, 1.0equiv) and 4-pyrazoleboronic acid pinacol ester (928 mg, 4.8 mmol, 1.1equiv) were taken up in MeCN (23 mL, 0.2 M) and Cs₂CO₃ (1.6 g, 4.8 mmol,1.1 equiv) was added. The resulting mixture was stirred at roomtemperature for 3 h. Upon completion, the mixture was diluted withCH₂Cl₂ (20 mL) and filtered through a fritted funnel. The filtrate wasconcentrated in vacuo to afford2-(6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)pyridin-2-yl)propan-2-olwhich was used in subsequent reactions without further purification.

Step 3: A solution of2-(6-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)methyl)pyridin-2-yl)propan-2-ol(127 mg, 0.37 mmol, 1.2 equiv) and 4-chloro-8-methoxyquinazolin-2-amine(71 mg, 0.34 mmol, 1.0 equiv) in MeCN (1 mL, 0.3 M) and 2.0 M aqueousK₂CO₃ (0.4 mL, 2.0 equiv) was sparged with N₂ for 10 minutes. Followingthis time, Pd(PPh₃)₄ (39 mg, 0.03 mmol, 0.1 equiv) was added and thereaction mixture heated to 100° C. for 14 h. Upon completion, thereaction mixture was filtered over celite, the filter cake washed withCH₂Cl₂ (10 mL), and the filtrate concentrated in vacuo. The brownresidue was purified by reversed-phase preparatory HPLC (95:5→5:95H₂O:MeCN with 0.1% CF₃CO₂H over 45 min) to give the title compound (45mg, 34% yield) as a yellow solid. ¹H NMR (400 MHz, Acetone-d₆) δ 8.92(s, 1H), 8.31 (s, 1H), 8.14-8.01 (m, 1H), 7.83 (t, J=7.8 Hz, 1H),7.68-7.45 (m, 3H), 7.18 (d, J=7.6 Hz, 1H), 5.67 (s, 2H), 4.08 (d, J=1.1Hz, 3H), 1.48 (d, J=1.1 Hz, 6H). LC-MS retention time 2.06 min; (M+H)⁺391.

Example 27 Synthesis of2-(6-{[3-(2-amino-8-methoxyquinazolin-4-yl)-1H-pyrazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 26.¹H NMR (400 MHz, DMSO-d₆) δ 8.49 (d, J=8.3 Hz, 1H), 8.05 (s, 1H),7.81-7.70 (m, 1H), 7.56 (d, J=7.9 Hz, 1H), 7.14-7.02 (m, 2H), 6.99 (d,J=2.3 Hz, 1H), 6.90 (d, J=7.7 Hz, 1H), 6.79 (s, 2H), 5.59 (s, 2H), 5.22(s, 1H), 3.85 (s, 3H), 1.41 (s, 6H). ESI MS [M+H]⁺ for C₂₁H₂₂N₆O₂, calcd391.2, found 391.2.

Example 28 Synthesis of2-(6-{[4-(2-aminoquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

Step 1: A solution of 2,4-dichloroquinoline (990 mg, 5 mmol, 1 equiv.),Pd₂dba₃ (115 mg, 0.13 mmol, 2.5 mol %), rac-BINAP (311 mg, 0.5 mmol, 10mol %), and sodium tert-butoxide (576 mg, 6 mmol, 1.2 equiv.) in toluene(20 mL) was stirred at room temperature under N₂ for approximately 5minutes. Benzophenone imine (0.84 mL, 5 mmol, 1 equiv.) was added, andthe reaction vial was placed in a heating block preheated to 100° C. forthree hours. The reaction mixture was cooled to room temperature, MTBEwas added, and the mixture was filtered through a short plug of Celiteand concentrated. The crude residue was dissolved in 25 mL THF, and 6 mLof 1M HCl was added. The reaction mixture stirred overnight and wasquenched by addition of saturated aqueous NaHCO₃, extracted with EtOAc,dried, and concentrated. The crude residue was purified by flashchromatography on SiO₂ (10-75% EtOAc/Hexanes) to afford2-amino-4-chloroquinoline (637 mg, 71%) as a tan solid.

Step 2: 2-amino-4-chloroquinoline (563 mg, 3.15 mmol, 1 equiv.),trimethylsilylacetylene (2.2 mL, 15.8 mmol, 5 equiv.), Cs₂CO₃ (3.1 g,9.5 mmol, 3 equiv.), Pd(OAc)₂ (35 mg, 0.16 mmol, 5 mol %),2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl (XPhos, 150 mg,0.32 mmol, 10 mol %), and dioxane (13 mL) were combined in a sealedvial. The vial was evacuated and backfilled with N₂ three times beforebeing placed in a heating block preheated to 100° C. for one hour. Themixture was filtered through a plug of Celite and concentrated. Thecrude residue (ca. 3.15 mmol) was suspended in MeOH (10 mL) and wasadded NH₃ solution (7 M in MeOH, 0.76 mL) at room temperature. After 45minutes, the reaction mixture was concentrated. The crude residue waspurified by flash chromatography on SiO₂ (50-100% EtOAc/CH₂Cl₂) toafford alkyne derivative (239 mg, 45%) as a brownish solid.

Step 3: Compound from step 2 (42 mg, 0.25 mmol, 1 equiv.) and azidederivative (from example 1, 48 mg, 0.25 mmol, 1 equiv.) were dissolvedin CH₂Cl₂ (0.5 mL) and a 2:1 mixture of tBuOH/H₂O (1 mL). CuSO₄·5H₂O(3.1 mg, 0.013 mmol, 5 mol %) and sodium ascorbate (9.9 mg, 0.05 mmol,20 mol %) were added, and the mixture was stirred until the startingmaterial was consumed as determined by LCMS analysis. The reactionmixture was directly purified by flash chromatography on SiO₂ (50-100%EtOAc/CH₂Cl₂) to afford the title compound (59 mg, 66%) as a tan solid.¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.82(t, J=7.8 Hz, 1H), 7.62 (d, J=7.8 Hz, 1H), 7.53 (s, 2H), 7.23-7.15 (m,2H), 7.09 (s, 1H), 6.59 (s, 2H), 5.83 (s, 2H), 5.24 (d, J=1.0 Hz, 1H),1.39 (d, J=2.2 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₂₀N₆O, calcd 361.2, found361.3.

Example 29 Synthesis of2-(6-{[4-(2-amino-8-methoxyquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

Step 1: To a solution of 4-chloro-8-methoxyquinoline (1 g, 5.16 mmol, 1equiv.) in CH₂Cl₂ (20 mL) was added m-CPBA (ca. 75%, 2.37 g, 10.3 mmol,2 equiv.). The reaction mixture stirred overnight and was quenched with10% aqueous KOH. The layers were separated, and the organic layer wasdried and concentrated to afford N-oxide derivative (796 mg, 74%) as anorange solid.

Step 2: A solution of the step 1 product (796 mg, 3.82 mmol, 1 equiv.)and tert-butylamine (2 mL, 19.1 mmol, 5 equiv.) in PhCF₃ (19 mL) wascooled in an ice/water bath, and Ts₂O (2.87 g, 8.8 mmol, 2.3 equiv.) wasadded in one portion. After 10 minutes, trifluoroacetic acid (9.6 mL,2.5 mL/mmol substrate) was added, and the reaction mixture was placed ina heating block preheated to 70° C. overnight. The reaction mixture wasconcentrated, and the residue was dissolved in CH₂Cl₂ and washed with10% aqueous KOH. The organic layer was concentrated, and the cruderesidue was purified by flash chromatography on SiO₂ (0-25% MeOH/CH₂Cl₂)to afford 2-amino-4-chloro-8-methoxyquinoline (390 mg, 49%) as a yellowsolid. Steps 3 and 4 were carried out in a similar fashion to example 28to afford 51 mg of the title compound. ¹H NMR (400 MHz, DMSO-d₆) δ 8.75(d, J=3.1 Hz, 1H), 7.82 (td, J=7.8, 3.1 Hz, 1H), 7.67 (d, J=8.3 Hz, 1H),7.65-7.57 (m, 1H), 7.21-7.13 (m, 1H), 7.13-7.00 (m, 3H), 6.62 (s, 2H),5.82 (d, J=2.9 Hz, 2H), 5.24 (d, J=2.9 Hz, 1H), 3.88 (d, J=3.0 Hz, 3H),1.39 (d, J=2.9 Hz, 6H). ESI MS [M+H]⁺ for C₂₁H₂₂N₆O₂, calcd 391.2, found391.3.

Example 30 Synthesis of2-(6-{[4-(2-amino-8-methylquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 29 toafford 60 mg of a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J=1.8Hz, 1H), 7.92 (d, J=8.4 Hz, 1H), 7.86-7.70 (m, 1H), 7.43 (dd, J=20.1,7.4 Hz, 2H), 7.18 (dd, J=10.0, 7.7 Hz, 2H), 7.10 (d, J=1.8 Hz, 1H), 5.80(d, J=1.7 Hz, 2H), 4.77 (s, 2H), 2.68 (s, 3H), 1.56 (d, J=1.8 Hz, 6H).ESI MS [M+H]⁺ for C₂₁H₂₂N₆O, calcd 375.2, found 375.4.

Example 31 Synthesis of2-(6-{[4-(2-amino-8-fluoroquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}pyridin-2-yl)propan-2-ol

The title compound was synthesized in a similar fashion to example 29 toafford 16 mg of a tan solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (d, J=1.7Hz, 1H), 8.00-7.91 (m, 1H), 7.86-7.77 (m, 1H), 7.61 (d, J=7.9 Hz, 1H),7.43-7.30 (m, 1H), 7.20-7.09 (m, 3H), 6.84 (s, 2H), 5.83 (s, 2H),5.28-5.20 (m, 1H), 1.39 (d, J=1.6 Hz, 6H). ESI MS [M+H]⁺ for C₂₀H₂₀FN₆O,calcd 379.2, found 379.3.

Example 322-(6-{[4-(2-Amino-7-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives to afford 61 mg of ayellow foam. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94 (dd, J=9.2, 1.4 Hz, 1H),8.78 (d, J=1.6 Hz, 1H), 7.81 (td, J=7.8, 1.5 Hz, 1H), 7.61 (dd, J=7.9,1.4 Hz, 1H), 7.14 (dd, J=7.7, 1.5 Hz, 1H), 6.90 (ddd, J=9.3, 2.7, 1.5Hz, 1H), 6.84 (dd, J=2.7, 1.3 Hz, 1H), 6.68 (s, 2H), 5.86 (s, 2H), 5.22(d, J=1.5 Hz, 1H), 3.88 (d, J=1.4 Hz, 3H), 1.37 (d, J=1.4 Hz, 6H). ESIMS [M+H]⁺ for C₂₀H₂₁N₇O₂, calcd 392.2, found 392.2.

Example 332-(6-{[4-(2-Amino-7,8-difluoro-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.03-8.94 (m, 1H), 8.87 (s, 1H), 7.81 (t, J=7.8 Hz, 1H), 7.62(d, J=7.9 Hz, 1H), 7.43-7.21 (m, 3H), 7.16 (d, J=7.6 Hz, 1H), 5.89 (s,2H), 5.23 (s, 1H), 1.37 (s, 6H). ESI MS [M+H]⁺ for C₁₉H₁₈F₂N₇O, calcd398.2, found 398.1.

Example 342-(6-{[4-(2-Amino-8-ethoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.81 (s, 1H), 8.54 (d, J=7.0 Hz, 1H), 7.81 (t, J=7.8 Hz, 1H),7.61 (d, J=7.9 Hz, 1H), 7.20-7.11 (m, 3H), 6.85 (s, 2H), 5.86 (s, 2H),5.23 (s, 1H), 4.15 (q, J=7.0 Hz, 2H), 1.42 (t, J=7.0 Hz, 3H), 1.38 (s,6H). ESI MS [M+H]⁺ for C₂₁H₂₄N₇O₂, calcd 406.2, found 406.2.

Example 352-Amino-4-(1-{[6-(1-hydroxy-1-methylethyl)-2-pyridyl]methyl}-1H-1,2,3-triazol-4-yl)-7-quinazolinecarbonitrile

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.24 (d, J=8.6 Hz, 1H), 8.89 (s, 1H), 7.97 (s, 1H), 7.81 (t,J=7.8 Hz, 1H), 7.61 (d, J=7.9 Hz, 1H), 7.57 (d, J=8.6 Hz, 1H), 7.22 (s,2H), 7.17 (d, J=7.6 Hz, 1H), 5.89 (s, 2H), 5.23 (s, 1H), 1.37 (s, 6H).ESI MS [M+H]⁺ for C₂₀H₁₉N₈O, calcd 387.2, found 387.4.

Example 362-(6-{[4-(2-Amino-6-fluoro-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.04 (s, 1H), 8.69 (d, J=7.3 Hz, 1H), 7.84 (t, J=7.8 Hz, 1H),7.71-7.58 (m, 2H), 7.23 (d, J=6.6 Hz, 1H), 5.95 (s, 2H), 4.08 (s, 3H),1.35 (s, 6H). ESI MS [M+H]⁺ for C₂₀H₂₁FN₇O₂, calcd 410.2, found 410.2.

Example 372-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-1,1,1-trifluoro-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.81 (s, 1H), 8.52 (m, 1H), 7.93 (t, J=7.8 Hz, 1H), 7.77-7.68(m, 1H), 7.33 (dd, J=7.7, 1.0 Hz, 1H), 7.18-7.07 (m, 2H), 6.83 (s, 2H),6.78-6.68 (m, 1H), 5.91 (s, 2H), 3.87 (s, 3H), 1.61 (s, 3H). ESI MS[M+H]⁺ for C₂₀H₁₈F₃N₇O₂, calcd 446.2, found 446.2.

Example 38(S)-2-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-1,1,1-trifluoro-2-propanol

Step 1: A round-bottom flask was charged with 20.0 g (66.2 mmol) ofcommercially available 2-bromo-pyridine derivative. To this flask wasadded 132 mL of dry THF and cooled to −78° C. under N₂ nBuLi 33 mL (2.5M in THF) was added dropwise to the reaction at −78° C. and stirred for30 min. Trifluoromethylacetone (11.9 mL, 132.3 mmol) was then added inone-portion and the reaction warmed to room temperature over 2 h (LCMSshows formation of the desired addition product). The reaction mixturewas cooled back to 0° C. and 100 mL saturated aqueous NH₄Cl was added toquench the reaction. The aqueous layer was extracted with EtOAc (2×100mL), dried over Na₂SO₄ and concentrated. The crude material was purifiedby silica gel chromatography to obtain the desired pyridine-diol (12.4g, 56%).

Step 2: A mixture of DMAP (391.0 mg, 3.2 mmol) and the product from step1 (670 mg, 2.0 mmol) was dissolved in 2.8 mL CH₂Cl₂. The mixture wascooled to 0° C. under N₂ and 4-nitrophenyl chloroformate (564.4 mg, 2.8mmol) was added. The mixture was stirred at that temperature for 10minutes and 3 h at room temperature. A light-yellow slurry is formed,which was then cooled to 0° C. and (R)-(+)-1-(1-naphthyl)ethyl amine(484.7, 4.0 mmol) was added and stirred at room temperature for 3 h. Thereaction mixture was washed with 1N NaOH (3×50 mL), dried over Na₂SO₄and concentrated. The crude material was purified by silica gelchromatography to obtain the desired diastereomers. Yield of topdiastereomer 230 mg (22%). Yield of bottom diastereomer 160 mg (15%).

Step 3: The top diastereomer from step 2 (200 mg, 0.38 mmol) wasdissolved in a mixture of THF (0.4 mL), MeOH (0.4 mL) and H₂O (0.4 mL).To this mixture was added LiOH (159.4 mg, 3.8 mmol) and heated at 45° C.for 10 h. Solvent was evaporated and crude material was purified bysilica gel chromatography to obtain pure diol 71 mg (84.4%).

Steps 4 and 5: The title compound was synthesized in similar fashion toexample 1 from the corresponding azide and alkyne derivatives. ¹H NMR(400 MHz, DMSO-d₆) δ 8.81 (s, 1H), 8.58-8.46 (m, 1H), 7.99-7.85 (m, 1H),7.73 (d, J=7.9 Hz, 1H), 7.33 (d, J=7.7 Hz, 1H), 7.21-7.08 (m, 2H), 6.83(s, 2H), 6.73 (s, 1H), 5.91 (s, 2H), 3.87 (s, 3H), 1.61 (s, 3H). ESI MS[M+H]⁺ for C₂₀H₁₈F₃N₇O₂, calcd 446.2, found 446.2.

Example 39(R)-2-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-1,1,1-trifluoro-2-propanol

The title compound was synthesized in similar fashion to example 38 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.81 (s, 1H), 8.58-8.46 (m, 1H), 7.99-7.85 (m, 1H), 7.73 (d,J=7.9 Hz, 1H), 7.33 (d, J=7.7 Hz, 1H), 7.21-7.08 (m, 2H), 6.83 (s, 2H),6.73 (s, 1H), 5.91 (s, 2H), 3.87 (s, 3H), 1.61 (s, 3H). ESI MS [M+H]⁺for C₂₀H₁₈F₃N₇O₂, calcd 446.2, found 446.2.

Example 403-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-3-oxetanol

The title compound was synthesized in similar fashion to example 21 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.85 (s, 1H), 8.53 (m, 1H), 7.90-7.82 (m, 1H), 7.56 (d, J=7.9Hz, 1H), 7.25 (d, J=7.7 Hz, 1H), 7.16 (m, 2H), 6.88 (m, 2H), 6.65 (s,1H), 5.94 (s, 2H), 4.83 (d, J=5.9 Hz, 2H), 4.60 (d, J=6.0 Hz, 5H), 3.87(s, 3H). ESI MS [M+H]⁺ for C₂₀H₁₉N₇O₃, calcd 406.2, found 406.1.

Example 413-(6-{[4-(2-Amino-8-fluoro-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-3-oxetanol

The title compound was synthesized in similar fashion to example 21 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.89 (d, J=1.7 Hz, 1H), 8.85 (d, J=8.6 Hz, 1H), 7.87 (td,J=7.8, 1.7 Hz, 1H), 7.59-7.50 (m, 2H), 7.27 (d, J=7.8 Hz, 1H), 7.21 (m,1H), 7.10 (s, 2H), 6.55 (s, 1H), 5.94 (d, J=1.7 Hz, 2H), 5.74 (s, 1H),4.84 (d, J=6.0 Hz, 2H), 4.59 (d, J=6.1 Hz, 2H). ESI MS [M+H]⁺ forC₁₉H₁₆FN₇O₂, calcd 394.1, found 394.1.

Example 423-(6-{[4-(2-Amino-7-fluoro-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-3-oxetanol

The title compound was synthesized in similar fashion to example 21 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.90-8.79 (m, 2H), 7.91-7.81 (m, 1H), 7.55 (d, J=7.9 Hz, 1H),7.27 (d, J=7.7 Hz, 1H), 7.17 (m, 1H), 7.05 (s, 2H), 6.55 (s, 1H), 5.94(s, 2H), 4.83 (d, J=6.1 Hz, 2H), 4.59 (d, J=6.1 Hz, 2H), 3.98 (s, 3H).ESI MS [M+H]⁺ for C₂₀H₁₈N₇O₃, calcd 424.2, found 424.2.

Example 434-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)tetrahydro-2H-pyran-4-ol

The title compound was synthesized in similar fashion to example 21 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.84 (s, 1H), 8.58-8.50 (m, 1H), 7.85 (t, J=7.8 Hz, 1H), 7.64(d, J=8.0 Hz, 1H), 7.21 (d, J=7.7 Hz, 1H), 7.17 (d, J=3.7 Hz, 2H), 6.86(s, 2H), 5.88 (s, 2H), 5.30 (s, 1H), 3.89 (s, 3H), 3.78-3.63 (m, 4H),2.21-2.04 (m, 2H), 1.40 (d, J=13.1 Hz, 2H). ESI MS [M+H]⁺ forC₂₂H₂₄N₇O₃, calcd 434.2, found 434.1.

Example 442-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-methylpropanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.80 (s, 1H), 8.59-8.53 (m, 1H), 7.75 (t, J=7.8, 1.8 Hz, 1H),7.37 (d, J=8.1 Hz, 1H), 7.21-7.15 (m, 2H), 7.12 (d, J=7.7 Hz, 1H), 6.84(s, 2H), 5.85 (s, 2H), 4.58 (t, J=5.5 Hz, 1H), 3.88 (s, 3H), 3.51-3.45(m, 2H), 1.19 (s, 6H). ESI MS [M+H]⁺ for C₂₁H₂₄N₇O₂, calcd 406.2, found406.2.

Example 452-(6-{[4-(2-Amino-8-fluoro-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-methylpropanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.93-8.83 (m, 2H), 7.76 (t, J=7.7 Hz, 1H), 7.61-7.53 (m, 1H),7.37 (d, J=8.0 Hz, 1H), 7.27-7.18 (m, 1H), 7.18-7.03 (m, 3H), 5.87 (s,2H), 4.58 (t, J=5.6 Hz, 1H), 3.47 (d, J=5.6 Hz, 2H), 1.18 (s, 6H). ESIMS [M+H]⁺ for C₂₀H₂₁FN₇O, calcd 394.2, found 394.2.

Example 462-(6-{[4-(2-Amino-7-fluoro-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-methylpropanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.87 (dd, J=9.2, 5.7 Hz, 1H), 8.83 (s, 1H), 7.76 (t, J=7.8Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.24-7.16 (m, 1H), 7.14 (d, J=8.0 Hz,1H), 7.07 (s, 2H), 5.87 (s, 2H), 4.58 (t, J=5.5 Hz, 1H), 4.00 (s, 3H),3.47 (d, J=5.5 Hz, 2H), 1.18 (s, 6H). ESI MS [M+H]⁺ for C₂₁H₂₃FN₇O₂,calcd 424.2, found 424.2.

Example 478-Chloro-4-[1-({6-[(2-methoxyethoxy)methyl]-2-pyridyl}methyl)-1H-1,2,3-triazol-4-yl]-2-quinazolinylamine

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.05 (d, J=8.4 Hz, 1H), 8.83 (s, 1H), 7.92-7.80 (m, 2H), 7.40(d, J=7.7 Hz, 1H), 7.30-7.20 (m, 2H), 7.16 (s, 2H), 5.86 (s, 2H), 4.53(s, 2H), 3.59 (ddd, J=6.0, 3.1, 1.0 Hz, 2H), 3.46 (ddd, J=6.4, 3.7, 1.0Hz, 2H), 3.21 (s, 3H). ESI MS [M+H]⁺ for C₂₀H₂₀ClN₇O₂, calcd 426.1,found 426.1.

Example 484-(1-{[6-({[(S)-Tetrahydrofur-2-yl]methoxy}methyl)-2-pyridyl]methyl}-1H-1,2,3-triazol-4-yl)-8-methoxy-2-quinazolinylamine

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives to afford 110 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86-8.72 (m, 1H), 8.69-8.50(m, 1H), 7.95-7.83 (m, 1H), 7.42 (d, J=7.7 Hz, 1H), 7.28 (d, J=7.6 Hz,1H), 7.18 (dd, J=4.8, 2.8 Hz, 2H), 6.86 (s, 2H), 5.86 (s, 2H), 4.56 (d,J=3.4 Hz, 2H), 4.00-3.92 (m, 1H), 3.89 (d, J=3.5 Hz, 3H), 3.74-3.67 (m,1H), 3.59 (q, J=7.9, 7.3 Hz, 1H), 3.50-3.42 (m, 1H), 1.85 (ddd, J=15.3,7.8, 3.6 Hz, 1H), 1.74 (q, J=7.0 Hz, 2H), 1.51 (dq, J=11.5, 7.5 Hz, 1H).ESI MS [M+H]⁺ for C₂₃H₂₄N₇O₃, calcd 448.2, found 448.2.

Example 494-(1-{[6-({[(R)-Tetrahydrofur-2-yl]methoxy}methyl)-2-pyridyl]methyl}-1H-1,2,3-triazol-4-yl)-8-methoxy-2-quinazolinylamine

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives to afford 72 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86-8.72 (m, 1H), 8.69-8.50(m, 1H), 7.95-7.83 (m, 1H), 7.42 (d, J=7.7 Hz, 1H), 7.28 (d, J=7.6 Hz,1H), 7.18 (dd, J=4.8, 2.8 Hz, 2H), 6.86 (s, 2H), 5.86 (s, 2H), 4.56 (d,J=3.4 Hz, 2H), 4.00-3.92 (m, 1H), 3.89 (d, J=3.5 Hz, 3H), 3.74-3.67 (m,1H), 3.59 (q, J=7.9, 7.3 Hz, 1H), 3.50-3.42 (m, 1H), 1.85 (ddd, J=15.3,7.8, 3.6 Hz, 1H), 1.74 (q, J=7.0 Hz, 2H), 1.51 (dq, J=11.5, 7.5 Hz, 1H).ESI MS [M+H]⁺ for C₂₃H₂₄N₇O₃, calcd 448.2, found 448.2.

Example 50 Ethyl4-(6-{[4-(2-amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-1-piperidinecarboxylate

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.81 (s, 1H), 8.60-8.52 (m, 1H), 7.78 (t, J=7.2 Hz, 1H), 7.29(d, J=7.8 Hz, 1H), 7.22-7.11 (m, 3H), 6.87 (s, 2H), 5.85 (s, 2H),4.15-3.96 (m, 4H), 3.89 (s, 3H), 3.02-2.75 (m, 3H), 1.87-1.74 (m, 2H),1.56 (qd, J=12.6, 4.2 Hz, 2H), 1.15 (t, J=7.1 Hz, 3H). ESI MS [M+H]⁺ forC₂₅H₂₉N₈O₃, calcd 489.2, found 489.2.

Example 511-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-3-pyrrolidinol

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.74 (s, 1H), 8.61-8.54 (m, 1H), 7.49 (t, J=7.8 Hz, 1H),7.21-7.13 (m, 2H), 6.85 (s, 2H), 6.47 (d, J=7.1 Hz, 1H), 6.39 (d, J=8.4Hz, 1H), 5.65 (s, 1H), 4.93 (d, J=3.6 Hz, 1H), 4.38-4.31 (m, 1H), 3.89(s, 3H), 3.46-3.35 (m, 3H), 3.25 (d, J=11.2 Hz, 1H), 2.04-1.91 (m, 1H),1.90-1.79 (m, 1H). ESI MS [M+H]⁺ for C₂₁H₂₃N₈O₂, calcd 419.2, found419.1.

Example 521-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)-2-pyrrolidinone

The title compound was synthesized in similar fashion to example 6 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.78 (d, J=1.4 Hz, 1H), 8.54 (ddd, J=5.6, 4.1, 1.6 Hz, 1H),8.25-8.20 (m, 1H), 7.84 (ddd, J=8.4, 7.3, 1.6 Hz, 1H), 7.21-7.13 (m,2H), 7.14-7.07 (m, 1H), 6.85 (s, 2H), 5.82 (s, 2H), 3.87 (s, 3H),3.85-3.78 (m, 2H), 2.57-2.50 (m, 2H), 2.03-1.86 (m, 2H). ESI MS [M+H]⁺for C₂₁H₂₀N₈O₂, calcd 417.2, found 417.1.

Example 53m-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)benzoicacid

Step 1: A solution of bromopyridine (376 mg, 2 mmol, 1 equiv.),arylboronic acid (432 mg, 2.4 mmol, 1.2 equiv.), and Na₂CO₃ (424 mg, 4mmol, 2 equiv.) in dioxane (4 mL) and H₂O (2 mL) was degassed with N₂for ca. 5 minutes, and Pd(dppf)Cl₂ (73 mg, 0.1 mmol, 5 mol %) was added.The reaction vessel was heated to 130° C. for two hours. The reactionmixture was cooled to room temperature, diluted with H₂O, and extractedwith EtOAc. The combined organic extracts were filtered through a plugof SiO₂, concentrated, and used without further purification.

Step 2: DBU (0.39 mL, 2.6 mmol, 1.3 equiv.) was added to a stirringsolution of substrate (ca. 2 mmol) and DPPA (0.56 mL, 2.6 mmol, 1.3equiv.) in PhMe (2.5 mL). The reaction mixture was stirred overnight,loaded directly onto SiO₂, and purified by flash chromatography on SiO₂to yield pyridine azide as a thick oil (411 mg).

Step 3: The ester was synthesized in a similar fashion to example 1 fromthe corresponding azide and alkyne derivatives to afford 145 mg of anorange foam.

Step 4: LiOH (1 M aqueous solution, 0.75 mL, 3 equiv.) was added to asuspension of triazole substrate (117 mg, 0.25 mmol, 1 equiv.) in THF(0.75 mL) at room temperature. After stirring overnight, 5 drops of AcOHwas added, and the reaction mixture was loaded directly onto SiO₂ andpurified by flash chromatography on SiO₂ to yield the title compound as113 mg of a yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (d, J=1.2Hz, 1H), 8.65 (d, J=1.6 Hz, 1H), 8.62-8.53 (m, 1H), 8.28 (d, J=7.8 Hz,1H), 8.08-7.90 (m, 4H), 7.61 (t, J=7.7 Hz, 1H), 7.34 (d, J=7.3 Hz, 1H),7.17 (d, J=4.7 Hz, 2H), 6.85 (s, 2H), 6.00 (s, 2H), 3.89 (d, J=1.1 Hz,3H). ESI MS [M+H]⁺ for C₂₄H₁₉N₇O₃, calcd 454.5, found 454.1.

Example 54o-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)benzoicacid

The title compound was synthesized in similar fashion to example 53 fromthe corresponding azide and alkyne derivatives to afford 50 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.79 (d, J=0.9 Hz, 1H), 8.54(dd, J=5.9, 3.8 Hz, 1H), 7.97-7.87 (m, 1H), 7.73 (d, J=7.5 Hz, 1H),7.63-7.48 (m, 5H), 7.29 (d, J=7.7 Hz, 1H), 7.20-7.11 (m, 2H), 6.85 (s,2H), 5.88 (s, 2H), 3.89 (d, J=0.8 Hz, 3H). ESI MS [M+H]⁺ for C₂₄H₁₉N₇O₃,calcd 454.5, found 454.1.

Example 55p-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-1,2,3-triazol-1-yl]methyl}-2-pyridyl)benzoicacid

The title compound was synthesized in similar fashion to example 53 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.11 (d, J=1.1 Hz, 1H), 8.89 (d, J=8.4 Hz, 1H), 8.12 (dd,J=8.6, 1.2 Hz, 2H), 8.07-7.95 (m, 4H), 7.58 (d, J=8.0 Hz, 1H), 7.49 (t,J=8.2 Hz, 1H), 7.44 (d, J=7.4 Hz, 1H), 6.06 (s, 2H), 4.01 (s, 3H). ESIMS [M+H]⁺ for C₂₄H₁₉N₇O₃, calcd 454.2, found 454.1.

Example 562-[6-({4-[2-(Cyclopropylamino)-8-methoxy-4-quinazolinyl]-1H-1,2,3-triazol-1-yl}methyl)-2-pyridyl]-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.85 (s, 1H), 8.60 (s, 1H), 7.80 (t, J=7.8 Hz, 1H), 7.60 (d,J=8.0 Hz, 1H), 7.18 (m, 2H), 5.85 (s, 2H), 5.21 (s, 1H), 3.89 (s, 3H),2.92 (m, 1H), 1.36 (s, 6H), 0.69 (m, 2H), 0.51 m, 2H). ESI MS [M+H]⁺ forC₂₃H₂₅N₇O₂, calcd 432.2, found 432.2.

Example 572-[6-({4-[2-(Isopropylamino)-8-methoxy-4-quinazolinyl]-1H-1,2,3-triazol-1-yl}methyl)-2-pyridyl]-2-propanol

The title compound was synthesized in similar fashion to example 1 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.87 (br s, 1H), 8.59 (br s, 1H), 7.82 (t, J=7.8 Hz, 1H),7.62 (d, J=8.0 Hz, 1H), 7.35-7.06 (m, 4H), 5.87 (s, 2H), 5.24 (s, 1H),4.37-4.20 (m, 1H), 3.90 (s, 3H), 1.38 (s, 6H), 1.20 (d, J=6.5 Hz, 6H).ESI MS [M+H]⁺ for C₂₃H₂₈N₇O₂, calcd 436.2, found 436.2.

Example 582-{[4-(2-Amino-8-fluoroquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(2-hydroxypropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives to afford 50 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86 (d, J=0.7 Hz, 1H),8.04-7.96 (m, 1H), 7.78-7.68 (m, 1H), 7.55-7.45 (m, 1H), 7.36 (ddd,J=11.2, 7.7, 1.3 Hz, 1H), 7.20-7.08 (m, 3H), 6.85 (s, 2H), 6.65 (s, 1H),5.94 (s, 2H), 1.61 (s, 6H). ESI MS [M+H]⁺ for C₂₀H₁₉FN₆O₂, calcd 395.2,found 395.1.

Example 592-{[4-(2-Amino-7-fluoro-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(2-hydroxypropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives to afford 48 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92 (d, J=1.7 Hz, 1H), 8.88(dd, J=9.3, 5.9 Hz, 1H), 7.72 (dd, J=8.1, 2.0 Hz, 1H), 7.49 (t, J=7.9Hz, 1H), 7.25-7.14 (m, 2H), 7.10 (s, 2H), 6.62 (d, J=1.7 Hz, 1H), 5.97(s, 2H), 4.00 (d, J=1.7 Hz, 3H), 1.60 (d, J=1.7 Hz, 6H). ESI MS [M+H]⁺for C₂₀H₂₀FN₇O₃, calcd 426.2, found 426.2.

Example 602-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-[(2S)-1,1,1-trifluoro-2-hydroxypropan-2-yl]pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.66 (s, 1H), 8.89 (s, 1H), 8.56 (s, 1H), 7.92 (d, J=8.1 Hz,1H), 7.64 (dd, J=8.0, 8.0 Hz, 1H), 7.33 (d, J=7.8 Hz, 1H), 7.17 (d,J=5.2 Hz, 2H), 6.87 (s, 2H), 6.00 (s, 2H), 3.88 (s, 3H), 1.80 (s, 3H).ESI MS [M+H]⁺ for C₂₀H₁₈F₃N₇O₃, calcd 462.1, found 462.2.

Example 612-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-[(2R)-1,1,1-trifluoro-2-hydroxypropan-2-yl[pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.66 (s, 1H), 8.89 (s, 1H), 8.56 (s, 1H), 7.92 (d, J=8.1 Hz,1H), 7.64 (dd, J=8.0, 8.0 Hz, 1H), 7.33 (d, J=7.8 Hz, 1H), 7.17 (d,J=5.2 Hz, 2H), 6.87 (s, 2H), 6.00 (s, 2H), 3.88 (s, 3H), 1.80 (s, 3H).ESI MS [M+H]⁺ for C₂₀H₁₈F₃N₇O₃, calcd 462.1, found 462.2.

Example 622-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

Step 1: A round-bottom flask was charged with 2.0 g (6.7 mmol) ofcommercially available 2-bromo-pyridine derivative. To this flask wasadded 13.0 mL of dry THF and cooled to −78° C. under N₂. nBuLi 2.7 mL(2.5 M in THF) was added dropwise to the reaction at −78° C. and stirredfor 30 min. Cyclobutanone (0.58 mL, 7.9 mmol) was then added inone-portion and the reaction warmed to room temperature over 2 h (LCMSshows formation of the desired addition product). The reaction mixturewas cooled back to 0° C. and 6.7 mL of TBAF (1 M in THF) was added.After stirring the reaction for 15 min at 0° C., 50.0 mL saturatedaqueous NH₄Cl was added to quench the reaction. The aqueous layer wasextracted with EtOAc (2×50 mL), dried over Na₂SO₄ and concentrated. Thecrude material was purified by silica gel chromatography to obtain thedesired pyridine-diol (570 mg, 48% in 2-steps).

Step 2: To a solution of the diol (570.0 mg, 3.2 mmol) from step 1 inCH₂Cl₂ (4.0 mL) was added diphenyl-phosphorylazide (0.8 mL, 3.8 mmol)and DBU (0.6 mL, 3.8 mmol) at room temperature. The reaction mixture wasstirred at room temperature for 10 h under N₂. After removing CH₂Cl₂,the residue was re-dissolved in EtOAc and subsequently washed with H₂O(2×25 mL). The organic layer was dried over Na₂SO₄ and concentrated. Thecrude material was purified by silica gel chromatography to obtain thedesired azide (450 mg, 69%).

Step 3: To a solution of azide (230 mg, 1.1 mmol) from step 2 in CH₂Cl₂(3.8 mL) was added 291.9 mg, 1.7 mmol of mCPBA (75% in water) at roomtemperature. The reaction mixture was stirred at room temperature for 3h under N₂. After removing CH₂Cl₂, the crude material was purified bysilica gel chromatography to obtain the desired N-oxide (235 mg, 97%).

Step 4: The title compound was synthesized similar to example 1. ¹H NMR(400 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.57 (m, 1H), 7.76-7.65 (m, 1H), 7.50(td, J=8.0, 1.4 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.19-7.12 (m, 2H),6.95-6.81 (m, 2H), 6.40 (s, 1H), 5.97 (s, 2H), 3.87 (s, 3H), 2.60-2.51(m, 2H), 2.30-2.16 (m, 2H), 2.00-1.87 (m, 1H), 1.71-1.55 (m, 1H). ESI MS[M+H]⁺ for C₂₁H₂₁N₇O₃, calcd 420.2, found 420.1

Example 632-{[4-(2-Amino-8-fluoroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.92 (d, J=1.5 Hz, 1H), 8.88 (d, J=8.5 Hz, 1H), 7.71 (d,J=8.0 Hz, 1H), 7.53 (m, 2H), 7.29-7.17 (m, 2H), 7.14 (s, 2H), 6.39 (s,1H), 5.98 (s, 2H), 5.74 (s, 1H), 2.61-2.50 (m, 2H), 2.30-2.17 (m, 2H),1.94 (m, 1H), 1.68-1.53 (m, 1H). ESI MS [M+H]⁺ for C₂₀H₁₈FN₇O₂, calcd408.2, found 408.1.

Example 642-{[4-(2-Amino-8-chlorooquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 9.04 (d, J=8.5 Hz, 1H), 8.92 (d, J=2.0 Hz, 1H), 7.88 (d,J=7.5 Hz, 1H), 7.71 (d, J=8.3 Hz, 1H), 7.50 (m, 1H), 7.28-7.14 (m, 4H),6.39 (s, 1H), 5.98 (s, 2H), 2.60-2.51 (m, 2H), 2.32-2.18 (m, 2H), 1.93(m, 1H), 1.63 (m, 1H). ESI MS [M+H]⁺ for C₂₀H₁₈ClN₇O₂, calcd 424.1,found 424.1.

Example 652-{[4-(2-Amino-8-methylquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives to afford 90 mg of awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.92-8.85 (m, 2H), 7.73 (dd,J=8.1, 1.9 Hz, 1H), 7.58 (ddd, J=7.0, 1.8, 1.0 Hz, 1H), 7.52 (td, J=7.9,1.4 Hz, 1H), 7.24 (dd, J=7.9, 1.9 Hz, 1H), 7.21-7.13 (m, 1H), 6.82 (s,2H), 6.42 (d, J=1.0 Hz, 1H), 5.98 (s, 2H), 2.56 (dt, J=8.3, 4.5 Hz, 2H),2.52 (s, 3H), 2.33-2.19 (m, 2H), 1.95 (dt, J=9.7, 4.9 Hz, 1H), 1.65 (p,J=8.8 Hz, 1H). ESI MS [M+H]⁺ for C₂₁H₂₁N₇O₂, calcd 404.2, found 404.2.

Example 662-{[4-(2-Amino-7-fluoro-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives to afford 48 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.94-8.85 (m, 2H), 7.73 (dd,J=8.0, 2.0 Hz, 1H), 7.58-7.45 (m, 1H), 7.26 (dd, J=7.9, 1.9 Hz, 1H),7.24-7.17 (m, 1H), 7.10 (s, 2H), 6.45-6.39 (m, 1H), 5.99 (s, 2H), 4.00(d, J=2.4 Hz, 3H), 2.62-2.53 (m, 2H), 2.32-2.17 (m, 2H), 2.03-1.84 (m,1H), 1.72-1.56 (m, 1H). ESI MS [M+H]⁺ for C₂₁H₂₀FN₇O₃, calcd 438.2,found 438.2.

Example 672-{[4-(2-Amino-6-fluoro-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) 8.92 (s, 1H), 8.41 (d, J=6.0 Hz, 1H), 7.73 (d, J=4 Hz, 1H),7.52 (t, J=4 Hz, 1H), 7.26 (d, J=4 Hz, 1H), 7.18 (d, J=6 Hz, 1H), 6.90(s, 2H), 6.41 (s, 1H), 5.99 (s, 2H), 3.93 (s, 3H), 2.63-2.56 (m, 2H),2.26 (d, J=6 Hz, 2H), 1.95 (s, 1H), 1.64 (s, 1H); ESI MS [M+H]⁺ forC₂₁H₂₀FN₇O₃, calcd 438.2, found 438.2.

Example 682-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(4-hydroxyoxan-4-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.90 (s, 1H), 8.63-8.56 (m, 1H), 7.73 (d, J=8.3 Hz, 1H), 7.55(t, J=7.9 Hz, 1H), 7.22 (d, J=8.2 Hz, 1H), 7.20-7.13 (m, 2H), 6.89 (s,2H), 6.80 (s, 1H), 5.97 (s, 2H), 3.88 (s, 3H), 3.85-3.69 (m, 4H),2.47-2.36 (m, 2H), 1.71 (d, J=12.9 Hz, 2H). ESI MS [M+H]⁺ forC₂₂H₂₄N₇O₄, calcd 450.2, found 450.1.

Example 692-{[4-(2-Amino-7-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(3-hydroxy-1,1-dioxo-1λ⁶-thietan-3-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 62 fromthe corresponding azide and alkyne derivatives to afford 9 mg of a whitesolid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (dd, J=9.3, 2.0 Hz, 1H), 8.85(d, J=1.9 Hz, 1H), 7.84 (d, J=8.1 Hz, 1H), 7.61-7.48 (m, 1H), 7.30 (d,J=7.6 Hz, 1H), 7.20 (d, J=1.9 Hz, 1H), 6.91 (dd, J=9.3, 2.4 Hz, 1H),6.85 (t, J=2.2 Hz, 1H), 6.72 (s, 2H), 5.98 (s, 2H), 5.27 (d, J=12.9 Hz,2H), 4.08-3.96 (m, 2H), 3.89 (d, J=2.0 Hz, 3H). ESI MS [M+H]⁺ forC₂₀H₁₉N₇O₅S, calcd 470.1, found 470.1.

Example 702-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxy-2-methylpropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,Chloroform-d) δ 8.89 (s, 1H), 8.63-8.55 (m, 1H), 7.50 (d, J=8.2 Hz, 1H),7.32 (t, J=7.9 Hz, 1H), 7.22-7.13 (m, 2H), 7.02 (d, J=7.8 Hz, 1H), 6.89(s, 2H), 5.89 (s, 2H), 4.79-4.72 (m, 1H), 3.88 (s, 3H), 3.84 (d, J=3.7Hz, 2H), 1.38 (s, 6H). ESI MS [M+H]⁺ for C₂₁H₂₄N₇O₃, calcd 422.2, found422.2.

Example 712-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-methoxy-2-methylpropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives. ¹H NMR (400 MHz,DMSO-d₆) δ 8.88 (s, 1H), 8.59 (dd, J=5.8, 4.0 Hz, 1H), 7.47 (d, J=8.3Hz, 1H), 7.37-7.30 (m, 1H), 7.22-7.14 (m, 2H), 7.09 (d, J=7.7 Hz, 1H),6.88 (s, 2H), 5.91 (s, 2H), 3.89 (s, 3H), 3.81 (s, 2H), 3.12 (s, 3H),1.41 (s, 6H). ESI MS [M+H]⁺ for C₂₂H₂₆N₇O₃, calcd 436.2, found 436.2.

Example 722-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-cyclopropylpyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives to afford 35 mg of ayellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.89 (d, J=1.9 Hz, 1H),8.67-8.54 (m, 1H), 7.34-7.22 (m, 1H), 7.17 (ddt, J=5.9, 4.2, 2.1 Hz,4H), 6.89 (s, 2H), 5.93 (s, 2H), 3.89 (d, J=2.0 Hz, 3H), 2.73-2.60 (m,1H), 1.08 (dq, J=7.6, 3.3, 2.7 Hz, 2H), 0.83 (tq, J=6.6, 4.2, 3.1 Hz,2H). ESI MS [M+H]⁺ for C₂₀H₁₉N₇O₂, calcd 390.2, found 390.2.

Example 732-{[4-(2-Amino-8-chloroquinazolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(cyclobut-1-en-1-yl)pyridin-1-ium-1-olate

¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (d, J=8.5 Hz, 1H), 8.88 (d, J=2.4 Hz,1H), 7.88 (d, J=7.5 Hz, 1H), 7.47-7.40 (m, 1H), 7.37 (m, 1H), 7.32-7.27(m, 1H), 7.26-7.14 (m, 3H), 7.09-7.03 (m, 1H), 5.93 (s, 2H), 2.85-2.78(m, 2H), 2.57 (s, 2H). ESI MS [M+H]⁺ for C₂₀H₁₆ClN₇O, calcd 406.1, found406.1.

Example 74 8-Methoxy-4-(1H-pyrazol-4-yl)-2-quinazolinylamine

Step 1: A mixture 2,4-dichloro-8-methoxyquinazoline (10 g, 43.65 mmol),4,4,5,5-tetramethyl-2-[1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl]-1,3,2-dioxaborolane(12.14 g, 43.65 mmol), Na₂CO₃ (13.88 g, 130.95 mmol) in toluene:acetonitrile: water (350 mL:44 mL:44 mL, 8:1:1) was degassed with N₂ gasfor 5 minutes, Pd(PPh₃)₄ (Strem, 2.52 g, 2.18 mmol, 5 mol %) was addedand the reaction mixture was heated at 105° C. (external temperature)under N₂ for 16 h. TLC indicated completion of the reaction(CH₂Cl₂:EtOAc 1:1), the organic layer was separated, aqueous phase wasdiluted with water (100 mL), and extracted with ethyl acetate (2×100mL). The combined organic layer was concentrated under vacuum, theresidue was dissolved in dichloromethane, and purified by flash column(ISCO, 330 g column, EtOAc in CH₂Cl₂, 0 to 20%) to afford the product(12 g, 80%).

Step 2: A round-bottom flask was charged with 45.5 g (131.9 mmol) of2-chloro-quinazoline derivative from step 1, p-methoxy benzylamine(103.4 mL, 1.3 mol) and K₂CO₃ 18.2 g (131.9 mmol). To this flask wasadded 445 mL of dry 2-Me-THF and heated to 80° C. The reaction wasstirred at this temperature under N₂ for 20 h. After cooling thereaction to room temperature, solid K₂CO₃ was filtered off. To thefiltrate was added 1.0 L 20% aqueous citric acid. The resultingprecipitate was filtered, washed with 200 mL water and dried. This driedcrude material was triturated with hot MTBE and taken to next stepwithout further purification.

Step 3: The crude product from step 2 was suspended in 187 mL TFA andheated at 70° C. for 15 h. After cooling to room temperature, 187 mL ofwater was added. The mixture was then basified to ≈ pH 10 using 10 NNaOH at 0° C. The yellow precipitate thus obtained was filtered. Theyellow precipitate of the product thus obtained contains some blackimpurity. To purify the product, the crude material was dissolved in 3MHCl, filtered off the black specks and basified the filtrate back to ≈pH 10 using 10 N NaOH at 0° C. The yellow precipitate thus obtained wasfiltered, washed with water and dried at 45° C. to obtain purepyrazolo-quinazoline (25.2 g, 79% over 2-steps).

Step 1: Aminobenzoic acid (154.5 g, 924 mmol) was dissolved in TFAA (924mL) at 0° C. with stirring. The reaction mixture was concentrated after30 minutes. Azeotropic removal of residual TFA was accomplished byadding toluene (300 mL) and concentrating the resulting mixture threetimes. The benzoxazinone product was isolated as 226 grams of a greyishsolid and used without further purification.

Step 2: Iodopyrazole (153 g, 550 mmol, 1.1 equiv.) was dissolved in THF(2 L) under N₂ and the solution was cooled in an ice-water bath.i-PrMgCl (2 M in THF, 300 mL, 600 mmol, 1.2 equiv.) was added dropwisewith vigorous stirring. After 60 minutes, solid benzoxazinone substratewas added, and the ice bath was removed. After 45 minutes, 10% aqueousNaOH (720 mL, 2 mol, 4 equiv.) was added, and the mixture was heated to65° C. After five hours, the reaction mixture was cooled to roomtemperature, quenched with 10% aqueous HCl (690 mL, 2 mol, 4 equiv.),and extracted with EtOAc. The combined organic extracts were dried andconcentrated to afford a viscous yellow/orange oil that was used withoutfurther purification.

Step 3: Amino-ketone (ca. 500 mmol) was dissolved in MeOH (1.5 L), andHCl (3M in MeOH, 500 mL, 3 equiv.) was added. The mixture was heated to65° C. After 60 minutes, the reaction mixture was cooled to roomtemperature and concentrated to afford a reddish solid. The residualsolid was suspended in iPrOH (1 L) and heated to reflux for ca. 1 hour.The suspension was cooled to room temperature and filtered. The solidwas washed with addition iPrOH and MTBE and dried to yield 69 grams of ayellowish solid. Note: Deprotection of the THP group occurs at ambienttemperature, and heating is optional to increase the rate ofdeprotection.

Step 4: NH₂CN (46.2 g, 7.0 equiv, 1100 mmol) was added to a mixture ofstep 3 product (40 g, 157.2 mmol) in acetic acid (310 mL) in a 2 L roundbottom flask at room temperature. Then the reaction mixture was heatedat 105° C. (external temperature) for 20 hours with stirring. It wascooled to room temperature, diluted with 4N aqueous HCl (310 mL), heatedat 105° C. (external temperature) for 24 hours. LCMS indicatedcompletion of the reaction, it was cooled to 0° C., carefullyneutralized with 8N aqueous NaOH solution (520 mL). The resulted yellowprecipitate was filtered, washed thoroughly with water (2×1000 mL), anddried under vacuum to give the title quinazoline (29 g, 76%).

Example 75 1-[6-(Chloromethyl)-2-pyridyl]cyclobutanol

Step 1: To THF (750 mL) at −78° C. was added n-butyllithium (100 mL, 250mmol, 2.5 M in hexanes). 2-bromo-6-methylpyridine (43.0 g, 250 mmol) wasthen added dropwise and the reaction mixture was stirred at −78° C. for10 minutes. Cyclobutanone (21.0 g; 300 mmol) was added dropwise, thereaction mixture was warmed to 0° C. and stirred for 1 hour. Sat. NH₄Cl(_(aq)) (100 mL) and brine (100 mL) were added. The organic phase wasdried over Na₂SO₄, concentrated, and subjected directly to the nextstep.

Step 2: To a solution of crude product from step 1 and CH₂Cl₂ (500 mL)at 0° C. was added m-CPBA (86.3 g, 375 mmol, 75% w/w). The reactionmixture was stirred at r.t. for 1 hour and 1 M NaOH(_(a)q) (500 mL) wasadded and the mixture stirred for 10 minutes. The organic phase wasseparated and the aqueous phase was extracted with CH₂Cl₂ (2×250 mL).The combined organic phases were dried over Na₂SO₄ and concentrated. Thecrude material (42.3 g) was recrystallized from MTBE (150 mL) to obtain28.1 g of pure material. The concentrated mother liquor (14.5 g) wassubjected to a second recrystallization with MTBE (50 mL) to obtain anadditional 5.01 g of pure material. Light brown crystals (33.13 g; 74%).

Step 3: To TFAA (1.35 L) at 0° C. was added the product from step 2 (230g, 1.28 mol) portionwise to keep the reaction mixture temperature below35° C. The reaction mixture was stirred at r.t. for 24 h, cooled to 0°C., and quenched with water (350 mL). The volatiles were removed bydistillation at ambient pressure in three separate batches. Distillationoccurs at 104° C. and is substantially complete when the headtemperature drops below 90° C. Water (1.2 L) was added and the aqueousphase washed twice with CH₂Cl₂ (1.2 L and 600 mL, respectively). Theaqueous phase was basified to pH=10 by addition of 20% NaOH_((aq)),extracted with 4:1 CH₂Cl₂:IPA (3×1.5 L), dried over Na₂SO₄, andconcentrated. The obtained oil was re-dissolved in EtOAc (900 mL),filtered through a silica plug, and concentrated to afford the desiredproduct as a dark yellow oil (188 g; 82%).

Step 4: To a suspension of N-chlorosuccinimide (63.2 g, 473 mmol) inCH₂Cl₂ (728 mL) at 0° C. was added a solution of triphenylphosphine (124g, 473 mmol) in CH₂Cl₂ (728 mL). The reaction mixture was stirred at 0°C. for 10 minutes and the product from step 3 (65.2 g, 364 mmol) inCH₂Cl₂ (182 mL) was added. The reaction mixture was stirred at r.t. for4 hours. The organic phase was extracted with 0.5 M HCl(_(a)q) (3×1.5L). The aqueous extracts were basified with 4 M NaOH to pH=7 andextracted with MTBE (1×1L). The organic phase was dried over Na₂SO₄ andpassed through a silica plug to afford the desired product as an orangeoil (63.2 g; 88%).

Example 761-(6-{[4-(2-Amino-8-methoxy-4-quinazolinyl)-1H-pyrazol-1-yl]methyl}-2-pyridyl)cyclobutanol

¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (s, 1H), 8.10 (s, 1H), 7.76 (t, J=7.7Hz, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.18-7.07 (m,2H), 7.01 (d, J=7.7 Hz, 1H), 6.76 (s, 2H), 5.75 (s, 1H), 5.59 (s, 2H),3.88 (s, 3H), 2.55-2.45 (m, 2H), 2.18 (q, J=9.0 Hz, 2H), 1.92-1.71 (m,2H). ESI MS [M+H]⁺ for C₂₂H₂₃N₆O₂, calcd 403.2, found 403.2.

Example 772-{[4-(2-Amino-8-methoxyquinazolin-4-yl)-1H-pyrazol-1-yl]methyl}-6-(1-hydroxycyclobutyl)pyridin-1-ium-1-olate

Step 1: To a solution of example 75 (63.2 g, 320 mmol) in CH₂Cl₂ (320mL) at 0° C. was added m-CPBA (77.2 g, 336 mmol, 75% w/w). The reactionmixture was stirred at r.t. for 3 hours and filtered through Na₂SO₄ toremove water. The organic phase was dry loaded onto silica gel andpurified by silica gel chromatography (0 to 100% EtOAc in hexanes). Thesolution was concentrated, redissolved in CH₂Cl₂ (1 L) and washed oncewith 0.1 M NaOH_((aq)) (1 L). The organic phase was dried over driedover Na₂SO₄ and concentrated to afford the desired product as a whitesolid (65.3 g, 96%).

Step 2: A round-bottom flask was charged with pyrazolo-quinazoline(example 74, 5.0 g, 20.7 mmol), chloromethyl-pyridine derivative (4.5 g,21.1 mmol) from the above step and K₂CO₃ (3.2 g, 22.8 mmol). To thismixture, 84 mL dry NMP was added and heated at 100° C. for 4 h. Aftercooling to room temperature, solid K₂CO₃ was filtered off. To thefiltrate was added 336 mL brine dropwise and stirred at room temperaturefor 1.5 h. The yellow precipitate thus obtained was filtered, washedwith 200 mL water and dried to obtain pure product 7.3 g (84%). ¹H NMR(400 MHz, DMSO-d₆) δ 8.70 (s, 1H), 8.17 (s, 1H), 7.68 (d, J=7.6 Hz, 2H),7.48 (t, J=7.9 Hz, 1H), 7.19-7.09 (m, 2H), 6.85 (d, J=8.0 Hz, 1H), 6.79(s, 2H), 6.54 (s, 1H), 5.69 (s, 2H), 3.88 (s, 3H), 2.63-2.53 (m, 2H),2.34-2.21 (m, 2H), 2.02-1.90 (m, 1H), 1.71-1.58 (m, 1H). ESI MS [M+H]⁺for C₂₂H₂₃N₆O₃, calcd 419.2, found 419.2.

Example 782-{[4-(2-Amino-8-fluoroquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(1-hydroxy-2-methylpropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives to afford 62 mg of awhite solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.85 (d, J=1.4 Hz, 1H), 7.99(d, J=8.4 Hz, 1H), 7.50 (d, J=8.1 Hz, 1H), 7.42-7.28 (m, 2H), 7.19-7.07(m, 3H), 6.98 (d, J=7.7 Hz, 1H), 6.84 (s, 2H), 5.87 (s, 2H), 3.86 (d,J=5.3 Hz, 2H), 1.48-1.31 (m, 6H). ESI MS [M+H]⁺ for C₂₁H₂₁FN₆O₂, calcd409.2, found 409.1.

Example 792-{[4-(2-Amino-8-methoxyquinolin-4-yl)-1H-1,2,3-triazol-1-yl]methyl}-6-(2-hydroxypropan-2-yl)pyridin-1-ium-1-olate

The title compound was synthesized in similar fashion to example 24 fromthe corresponding azide and alkyne derivatives to afford 60 mg of a tansolid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.81 (d, J=1.8 Hz, 1H), 7.71 (dd,J=9.3, 7.4 Hz, 2H), 7.53-7.45 (m, 1H), 7.15-7.06 (m, 3H), 7.03 (d, J=7.7Hz, 1H), 6.65 (d, J=1.7 Hz, 1H), 6.60 (s, 2H), 5.93 (s, 2H), 3.87 (d,J=1.7 Hz, 3H), 1.61 (d, J=1.8 Hz, 6H). ESI MS [M+H]⁺ for C₂₁H₂₂N₆O₃,calcd 407.2, found 407.2.

Analytical Methods:

LC: Agilent 1100 series; Mass spectrometer: Agilent G6120BA, single quad

LC-MS method: Agilent Zorbax Eclipse Plus C18 , 4.6×100 mm, 3.5 μM, 35°C., 1.5 mL/min flow rate, a 2.5 min gradient of 0% to 100% B with 0.5min wash at 100% B; A=0.1% of formic acid/5% acetonitrile/94.9% water;B=0.1% of formic acid/5% water/94.9% acetonitrile

Flash column: ISCO Rf+

Reverse phase HPLC: ISCO-EZ or Agilent 1260; Column: Kinetex 5 μm EVOC18 100 A; 250×21.2 mm (Phenomenex)

Biological Example Measurement of the Adenosine Receptor Activity ofCompounds using a human 2A Adenosine Receptor (A_(2a)R) CHO-TREx cAMPFunctional Assay

The cAMP antagonist functional assay (Perkin Elmer) was performed onCHO-T-REx cells induced to express Human A_(2A)R. Cells were seeded to awhite 384-well Opti plate at a density of 1,000 to 2,500 cells per wellfollowed by incubation with various concentrations of compound (rangingfrom 1 μM to 0 μM) at 37° C. for 1 hour.

A 1:2 serial dilution of NECA (Sigma Aldrich) from 1 μM to 0 μM wasadded to the cell stimulation mixture and incubated for 30 min at 37° C.After 30 min incubation, 5 μL of Ulight-anti-cAMP (1:150 dilution withconjugate and lysis buffer provided by Perkin Elmer) and 5 μL of Eu-cAMPtracer (1:50 dilution with conjugate and lysis buffer provided by PerkinElmer) was added the cells and incubated for an hour. FRET signal wasdetected with an Envision multilabel plate reader (Perkin Elmer) loadedwith 615 nm excitation filters and 665 nm emission filters.

Data analysis was performed using on GraphPad Prism (version 7.02) todetermine the K_(B) of the test compounds.

Measurement of the Adenosine Receptor Activity of Compounds Using aHuman 2B Adenosine Receptor CHO-K1 cAMP Functional Assay

CHO-K1 cells stably expressing Human Adenosine 2B Receptor (A_(2B)R:Cat. No. M000329, GenScript) were purchased from GenScript Inc.,Piscataway, N.J. 08854, USA.

The cAMP antagonist functional assay (Perkin Elmer) was performed onCHO-K1 cells stably expressing Human A_(2B)R. 1×10⁶ cells were seeded inT75 flasks and cultured at 37° C. and 5% CO₂ overnight. 2,000-4,000cells/well of stably-expressed A_(2b)R CHO-K1 cells were then seeded toa white 384-well Opti plate followed by compound 1 incubation at variousconcentrations (ranging from 1 μM to 0 μM) at 37° C. for 1 hour.

A 1:2 serial dilution of NECA (Sigma Aldrich) from 1 μM to 0 μM wasadded to the cell stimulation mixture and incubated for 30 min at 37° C.After 30 min incubation, 5 μL of Ulight-anti-cAMP (1:150 dilution withconjugate and lysis buffer provided by Perkin Elmer) and 5 μL of Eu-cAMPtracer (1:50 dilution with conjugate and lysis buffer provided by PerkinElmer) was added the cells and incubated for an hour. FRET signal wasdetected with an Envision multilabel plate reader (Perkin Elmer) loadedwith 615 nm excitation filters and 665 nm emission filters.

Data analysis was performed using on GraphPad Prism (version 7.02) todetermine the K_(B) of the test compounds.

TABLE 1 Specific Examples (Potency: A_(2A)R and A_(2B)R K_(B): +means >1 μM, ++ means 100 nM to 1 μM, +++ means <100 nM)

+++ +++

+++ +++

+++ +++

+++ ++

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+++ +++

+++ ++

+++ ++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ ++

+++ +++

+++ +++

+++ +++

+++ +++

+++ ++

+++ +++

+++ +++

+++ +++

+++ ++

+++ +++

+++ +++

++ ++

+++ ++

+++ +++

+++ ++

+++ +++

+++ +++

+++ ++

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+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

+++ +++

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A compound represented by Formula (I)

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein, the subscript m is 0 or 1, indicating a pyridine when m is 0 ora pyridine N-oxide when m is 1; G¹ is N or CR^(3a); G² is N or CR^(3b);R^(3a) and R^(3b) are each independently H or C₁₋₃ alkyl; R^(1a) andR^(1b) are each independently selected from the group consisting of i)H, ii) C₁₋₈ alkyl optionally substituted with from 1-3 R⁵ substituents,iii) —X¹—O—C₁₋₈ alkyl optionally substituted with from 1-3 R⁵substituents, iv) —C(O)—R⁶, v) Y optionally substituted with 1-3 R⁷substituents, vi) —X^(1a)—Y optionally substituted with 1-3 R⁷substituents; and vii) R^(1a) and R^(1b) together with the nitrogen towhich they are attached form a 5-6 membered heterocycloalkyl ringoptionally substituted with from 1-3 R⁸ substituents, wherein theheterocycloalkyl has 0-2 additional heteroatom ring vertices selectedfrom the group consisting of O, N, and S; each Y is C₃₋₈ cycloalkyl or 4to 6-membered heterocycloalkyl having 1-3 heteroatom ring verticesselected from the group consisting of O, N, and S; R² and R⁴ are eachindependently H or C₁₋₃ alkyl; each X¹ is C₁₋₆ alkylene; each R⁵ isindependently selected from the group consisting of hydroxyl, C₃₋₈cycloalkyl, phenyl, —O-phenyl, —C(O)OR^(a) and oxo; each R⁶ is C₁₋₈alkyl or Y, each of which is optionally substituted with 1-3substituents selected from the group consisting of hydroxyl, —O-phenyl,phenyl, and —O—C₁₋₈ alkyl; each R⁷ is independently selected from thegroup consisting of C₁₋₈ alkyl, hydroxyl, —O—C₁₋₈ alkyl, oxo, andC(O)OR^(a); each R⁸ is independently selected from the group consistingof C₁₋₈ alkyl, hydroxyl, and oxo; the subscript n is 0, 1, 2 or 3; eachR⁹ is independently selected from the group consisting of C₁₋₈ alkyl,C₁₋₈ haloalkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl,—X¹—O—X¹—O—C₁₋₈ alkyl, —C(O)OR^(a), halogen, cyano, phenyl,—NR^(b)R^(c), Y, —X¹—C₃₋₈ cycloalkyl, and —X²—Z, wherein X² is selectedfrom the group consisting of C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C₁₋₄alkylene-O—C₁₋₄ alkylene-, —C(O)—, and S(O)₂—, Z is 4 to 6-memberedheterocycloalkyl having 1-3 heteroatom ring vertices selected from thegroup consisting of O, N, and S, and wherein each of said R⁹substituents is optionally substituted with 1-3 R¹¹; each of R^(10a),R^(10b), R^(10c) and R^(10d) is independently selected from the groupconsisting of H, C₁₋₈ alkyl, halo, cyano, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈alkyl, —O—X¹—O—C₁₋₈ alkyl, —S(O)₂-C₁₋₆ alkyl, and —C(O)NR^(d)R^(e),wherein each of said R^(10a-d) substituents is optionally substitutedwith 1-3 R¹²; each R¹¹ is independently selected from the groupconsisting of hydroxyl, oxo, halo, cyano, —NR^(d)R^(e), —C(O)OR^(a),phenyl, C₃₋₈ cycloalkyl, and C₁₋₄ alkyl optionally substituted withC(O)OR^(a); each R¹² is independently selected from the group consistingof halo, cyano, hydroxy, —C(O)OR^(a); and each R^(a) is H or C₁₋₆ alkyl;each R^(b) and R^(c) are independently selected from the groupconsisting of H, C₁₋₈ alkyl, —S(O)₂—C₁₋₆ alkyl, —C(O)OR^(a), and—X¹—C(O)OR^(a); and each R^(d) and R^(e) are independently selected fromthe group consisting of H, C₁₋₈ alkyl, and —S(O)₂—C₁₋₆ alkyl.
 2. Thecompound of claim 1, wherein each R⁹ is independently selected from thegroup consisting of C₁₋₈ alkyl, —O—C₁₋₈ alkyl, —X¹—O—C₁₋₈ alkyl,—O—X¹—O—C₁₋₈ alkyl, —X¹—O—X¹—O—C₁₋₈ alkyl, —C(O)OR^(a), halogen, cyano,—NR^(b)R^(c), Y, —X¹—C₃₋₈ cycloalkyl, and —X²—Z, wherein X² is selectedfrom the group consisting of C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C₁₋₄alkylene-O—C₁₋₄ alkylene-, —C(O)—, and S(O)₂—, Z is 4 to 6-memberedheterocycloalkyl having 1-3 heteroatom ring vertices selected from thegroup consisting of O, N, and S, and wherein each of said R⁹substituents is optionally substituted with 1-3 R¹¹.
 3. The compound ofclaim 1, having Formula (Ia):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 4.The compound of claim 1, having Formula (Ib):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 5.The compound of any one of claims 1-4, wherein at least one of R^(10a),R^(10b), R^(10c) and R^(10d) is methoxy.
 6. The compound claim 1, havingFormula (Ic):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 7.The compound of claim 1, having Formula (Id):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 8.The compound of any of claims 1-7, or a pharmaceutically acceptablesalt, hydrate, or solvate thereof, wherein each R⁹ is independentlyselected from the group consisting of C₁₋₈ alkyl, —O—C₁₋₈ alkyl,—X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl, —X¹—O—X¹—O—C₁₋₈ alkyl, whereineach of said R⁹ substituents is optionally substituted with 1-3 R¹¹. 9.The compound of any of claims 1-7, or a pharmaceutically acceptablesalt, hydrate, or solvate thereof, wherein each R⁹ is independentlyselected from the group consisting of —C(O)OR^(a), —NR^(b)R^(c), Y,—X¹—C₃₋₈ cycloalkyl, and —X²—Z, wherein X² is selected from the groupconsisting of C₁₋₆ alkylene, —C₁₋₆ alkylene-O—, —C(O)—, and S(O)₂—, Z is4 to 6-membered heterocycloalkyl having 1-3 heteroatom ring verticesselected from the group consisting of O, N, and S, and wherein each ofsaid R⁹ substituents is optionally substituted with 1-3 R¹¹.
 10. Thecompound of claim 1, having Formula (Ie):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 11.The compound of claim 1, having Formula (If):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 12.The compound of claim 1, having Formula (Ig):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 13.The compound of claim 1, having Formula (Ih):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 14.The compound of claim 1, having Formula (Ii):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 15.The compound of claim 1, having Formula (Ij):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 16.The compound of claim 1, having Formula (Ik):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof ,wherein each R^(x) is independently C₁-C₃ alkyl, and optionally the twoR^(x) groups are joined together to form a 4, 5, or 6-membered ring. 17.The compound of claim 1, having Formula (Il):

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein each of R^(10a), R^(10b) and R^(10c) is independently selectedfrom the group consisting of C₁₋₈ alkyl, halo, cyano, —O—C₁₋₈ alkyl,—X¹—O—C₁₋₈ alkyl, —O—X¹—O—C₁₋₈ alkyl, and wherein each of said R^(10a),R^(10b) and R^(10c) is optionally substituted with 1-3 R¹².
 18. Thecompound of claim 1, selected from the group consisting of

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 19.A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable excipient.
 20. A compound selected from thegroup of